ES2556272T3 - Constructs of multicistronic siRNA to inhibit tumors - Google Patents

Abstract

A bicistronic short interference RNA construct comprising a first autocomplementary receptor sequence of the urokinase plasminogen activator (uPAR) and a second autocomplementary sequence of matrix metalloprotease type 9 (MMP-9) and an intervention sequence between the first and second autocomplementary sequences of approximately 22-68 base pairs.

Description

image 1

DESCRIPTION

Constructs of multicistronic siRNA to inhibit tumors

5 Background

Tumor progression involves modulation of tumor cell adhesion during migration and cellular degradation of the extracellular matrix (ECM) during tissue invasion. A complex balance of proteases, their activators and their inhibitors, regulates both processes during tumor invasion. Serine proteinases, metalloproteases and cysteine proteinases are three classes of proteinases that degrade ECM. The urokinase plasminogen activator (uPA) initiates a cascade of proteases that can degrade most of the matrix and basement membrane components and interfere with cell-cell and cell-matrix interactions. The uPA, together with its cell surface receptor, the urokinase plasminogen activator receptor (uPAR), is a participant in the degradation of the ECM, as demonstrated by an increased activation of the

15 plasminogen several times, uPA also activates several growth factors after degradation of ECM components. The union of uPA with its uPAR receptor activates downstream signaling molecules through a series of pathways, which include mitogen-activated protein kinases (MAPK) and signal transducer transducer and activator (Stat).

The recent discernment of interfering RNA (RNAi) has opened new avenues in cancer therapy. The RNAi is a mechanism of genetic silencing post-transcription, sequence specific that is influenced by double stranded RNA molecules homologous to the sequence of the target gene.

Interference RNA (RNAi) is a specific transcriptional genetic silencing mechanism.

25 sequence, which is activated with double stranded RNA (rRNAs) and causes degradation of mRNA with a sequence homologous to that of rRNA. RNAi depends on the formation of double stranded RNA (RNAds) whose antisense strand is complementary to that of the transcript of a targeted gene. Inhibition of sequence-specific RNAi can also be induced in mammalian cells. In one embodiment of RNAi, selective degradation of target mRNAs in mammalian cells was achieved by transfection with short, double-stranded interfering RNAs (siRNAs), which leads to rapid and efficient degradation of the target. It was shown that these siRNAs avoided nonspecific, well-documented effects triggered by longer double stranded RNAs in mammalian cells.

Prostate cancer is the second most common neoplasm in American men, with estimates of 230,110

35 new cases and approximately 30,000 deaths in 2004. As such, prostate cancer poses a major public health problem in the United States and around the world. At present, metastatic prostate cancer is incurable and ultimately claims the life of patients. A factor in the relative severity of prostate cancer is the ability to invade the component tumor cells that cause metastases. The invasive nature of tumor cells is a characteristic of cancer metastasis. Invasion of tumor cells and metastases are complex processes with three important stages: adhesion of malignant (neoplastic) cells to the extracellular matrix, digestion of the matrix to release cells from the primary tumor mass, and migration of tumor cells to targets high schools.

Glioblastoma multiforme (GBM) is a highly malignant primary central nervous system neoplasm, which is

45 highly resistant to therapy. One property that makes glioblastoma resistant to treatment is the tendency of tumor cells to invade normal brain tissue. Therapy that affects normal brain tissue is not acceptable. Therefore, invasion capacity is considered to be a major determinant of the malignant behavior of human gliomas. The invasion of a single diffuse cell, which occurs in all tumors of the glia regardless of histological grade, is defined as a translocation of neoplastic cells through cellular barriers and host ECM. Malignant gliomas express higher levels of uPA, uPAR and MMP-9 compared to normal brain tissue.

MMPs increase the invasion of tumor cells by degrading extracellular matrix proteins, activating signal transduction cascades that stimulate motility and activate growth factors, such

55 as transforming growth factor β, which are involved in the motility of GBM. The expression of MMP-2 and MMP-9 gelatinases correlates with the invasive and metastatic potentials of various cancers, including gliomas. MMP-9 levels correlated largely with the histological grade of the malignancy of the malignant disorder, glioma. MMP-9 is relevant in endothelial cell morphogenesis and capillary formation in glial / endothelial co-cultures in vitro. MMPs also regulate tumor angiogenesis and may be necessary for the 'angiogenic exchange' that occurs during tumor neovascularization.

The proteolytic activity of cathepsin B, a cysteine protease, involves the direct degradation of ECM proteins, including fibronectin, type I and IV collagen and laminin. Cathepsin B also indirectly activates other enzymes involved in the proteolytic cascade that mediates ECM degradation, including 65 soluble metalloproteinase and receptor-linked urokinase plasminogen activator (uPA). In addition, it has been suggested that cathepsin B increases MMP activity by inactivating tissue inhibitors of

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matrix metalloproteinases (TIMP). Therefore, cathepsin B could be an important upstream regulator in the activation of pro-uPA / plasminogen and pro-MMP. It has also been shown that Cathepsin B contributes to apoptosis causing the release of cytochrome c and activation of caspase 9 and 3 (fundamental events in the mitochondrial route of apoptosis). Increased expression of cathepsin B and reductions in its levels

5 inhibitors were associated with tumor growth, vascularization, invasion and metastasis in various cancers.

The development of siRNA molecules that target a plurality of genes involved in tumors to develop therapeutic compositions is desired.

Lakka S: et al., PROCEEDINGS OF THE ANNUAL MEETING OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 44, July 1, 2003 (), page 650, XP001525366, ISSN: 0197-016X is a summary of the meeting regarding the effects of "degradation of target RNA mediated by siRNA of uPAR and MMP-9 in cell lines of human glioma. " LAKKA SAJANI S ET AL .: "Synergistic down-regulation of urokinase plasminogen activator receptor and matrix

15 metalloproteinase-9 in SNB19 glioblastoma cells efficiently inhibits glioma cell invasion, angiogenesis, and tumor growth. ", CANCER RESEARCH, vol. 63, No. 10, May 15, 2003 (), pages 2454-2461, XP002608814, ISSN: 0008 -5472) refers to the efficacy of adenovirus-mediated expression of uPAR and MMP-9 antisense in inhibiting tumor invasion and growth in vitro and in vivo. CHRISTOPHER S GONDI ET AL: "Downregulation of uPA, uPAR and MMP -9 using small, interfering, hairpin RNA (siRNA) inhibits glioma cell invasion, angiogenesis and tumor growth "NEURON GLIA BIOLOGY ,, vol. 1, nº 2, October 25, 2004 (), page 176, XP002543013 ISSN: 1740- 925X doi: 1 0.1017 / S 1740925X04000237 describes a tricistronic construct and describes that MMP-9, uPAR and uPA play a role in tumor invasion and growth of glioma.

Summary

The invention is defined in the claims.

The invention relates to a bicistronic short-interference RNA construct comprising a first autocomplementary sequence of urokinase type plasminogen activator receptor (uPAR) and a second autocomplementary sequence of type 9 matrix metalloprotease (MMP-9) and a intervention sequence between the first and second autocomplementary sequences of approximately 22-68 base pairs.

The first autocomplementary sequence includes a nucleotide sequence of the urokinase plasminogen activator receptor (uPAR) and the second autocomplementary sequence comprises a sequence of

35 nucleotides of type 9 matrix metalloprotease (MMP-9). An autocomplementary sequence of uPAR is CTACAGCAGTGGAGAGCGATT-loop-AATCGCTCTCCACTGCTGTAG and an autocomplementary sequence of MMP-9 is CAAGTGGCACCACCACAACAA-loop-TTGTTGTGGT-GGTGCCACTTG in nucleus and the loop includes approximately 9 nuclei. A suitable loop sequence includes an ATATATAAT nucleotide sequence. The autocomplementary sequences of uPAR and MMP-9 are usually separated with an intervention sequence with a length of approximately 22-35 base pairs. An intervention sequence is GATCCA CTAGTAACGG CCGCCAGTGT GCTGG AATT. The uPAR-MMP-9 construct is a circular or a linear nucleic acid.

An autocomplementary sequence of uPAR is CTACAGCAGTGGAGAGCGATT-loop-AATCGCTCTCCACT

45 GCTGTAG; and an autocomplementary sequence of MMP-9 is CAAGTGGCACCACCACAACAA-loop-TTGTTGTGGT-GGTGCCACTTG. The loop includes approximately 9 nucleotides that are GC deficient. The loop sequence includes an ATATATAAT nucleotide sequence. The autocomplementary sequences of uPAR and MMP-9 are usually separated with an intervention sequence with a length of approximately 22-68 base pairs. An intervention sequence is GATCCA CTAGTAACGG CCGCCAGT-GT GCTGG AATTC TGCAGATATC CATCACACTG GCGGCCGC TCGA.

A method for inhibiting tumors is disclosed, a method that includes the steps of:

(a) administer a multicistronic short interference RNA construct; and 55

(b)

reduce the expression of a plurality of genes expressed in tumors, thereby inhibiting the formation

or tumor growth, and the reversal of tumors that already exist.

A method for using a multicistronic short-forming or interfering RNA construct that is directed, for example, to the urokinase-type plasminogen activator (uPAR) receptor and the urokinase-type plasminogen activator (uPA) is disclosed, reducing this way the expression of uPAR and uPA and inhibiting tumors. The multicistronic short-interference RNA construct includes a TGAGAGCCCTGCTGGCGCGCC-loop-GGCGCGCCAGCAGGGCTCTCA-intervention sequence-CTACAGCAGTGGAGAGCGATT-loop-AATCGCTCTCCACTGCTGTAG nucleotide sequence. Another expression for "65 intervention sequence" is a "spacer." A tumor is inhibited by reducing at least one of tumor cell proliferation, tumor cell invasion, tumor cell migration and angiogenesis. Some tumors

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They include prostate cancer, glioma, breast cancer, and melanoma. The construct is administered through a viral vector or administered by direct administration or by any suitable method known to those skilled in the art.

5 A method for using a multicistronic short-interference RNA construct can also be directed to the urokinase type plasminogen activator receptor (uPAR) and to the type 9 matrix metalloprotease (MMP-9), thereby reducing the expression of uPAR and MMP-9 and inhibiting tumors. The multicistronic short-interference RNA construct includes a CTACAG-CAGTGGAGAGCGATT-loop-AATCGCTCTCCACTGCTGTAG-spacer-CAAGTGGCACCACCACAACAA-loop-TT-GTTGTGGTGGTGCCACTTG nucleotide sequence. The tumor is inhibited by reducing at least one of tumor cell proliferation, tumor cell invasion, tumor cell migration and angiogenesis. Some tumors include prostate cancer, glioma, breast cancer, and melanoma. The construct is administered through a viral vector or administered by direct administration or by any suitable method known to those skilled in the art.

15 A method for inhibiting tumors is revealed, including the steps of:

(to)

administer a multicistronic construct directed to at least one of uPA, uPAR, MMP-9 and CB; Y

(b)

reduce the expression of at least one of uPA, uPAR, MMP-9 and CB, thereby inhibiting tumors.

A short interfering RNA molecule includes RNA molecules targeting:

(a) urokinase type plasminogen activator receptor (uPAR) and type 9 matrix metalloprotease (MMP-9), which includes a nucleic acid sequence CUACAGCAGUGGAGAGCGAUU-loop-AAUCGCUCUCCACUGCUGUAG-spacer-CAAGUGGCACCACCACAACAA-loop

25 UUGUUGUGGUGGUGCCACUUG.

A recombinant cell transformed with the uPAR-MMP-9 construct that is claimed is disclosed herein.

A recombinant virus transformed with the uPAR-MMP-9 construct that is claimed is disclosed herein.

Abbreviations

35 uPA refers to urokinase type plasminogen activator; uPAR refers to a urokinase type plasminogen activator receptor; MMP-9 refers to type 9 matrix metalloprotease; CB refers to cathepsin B; CMV refers to cytomegalovirus; SV40 refers to type 40 of ape virus; GFP refers to green fluorescent protein; NDE refers to extracellular matrix; SiRNA refers to short interference RNA; RNAsh refers to short hairpin RNA; RNAi refers to RNA interference.

Brief description of the figures

FIG. 1 shows that the expression levels of the uPA and uPAR proteins and the activity of uPA correlate with the invasive potential of human prostate cancer cell lines. The expression

The endogenous uPA and uPAR proteins were examined with immunoblot analysis of total cellular protein isolated from the following prostate cancer cell lines: LNCaP, DU145 and PC3. Equal amounts of protein isolated from cell extracts of the three cell lines were immunoblotted with anti-uPA, anti-uPAR and anti-GAPDH antibodies. GAPDH was used as a load control (A). The activity of uPA in prostate cancer cell lines was evaluated with fibrin zymography. Equal amounts of prostate cancer cell protein in serum-free media were separated with SDS-PAGE in 10% gels containing fibrinogen and plasminogen under non-reducing conditions. After exchange of SDS with washing with Triton X-100, the gel was incubated in glycine buffer (0.1 M, pH 8.0). Fibrinolytic activity was detected in the form of transparent lysis bands after staining with amido black and subsequent removal of staining with methanol-acetic acid (B).

55 Comparison of the in vitro invasive potentials of prostate cancer cell lines (C). Invasion assays were performed in 12 possible Transwell chambers containing polycarbonate filters with 12 µm pores coated with Matrigel. The cells that had been passed to the lower surface of the filters were stained and photographs taken under a microscope with a magnification of 200X (C). The cell invasion was counted through the Matrigel with a microscope in three random fields with an increase of 200X. Each / represents the mean ± SD of the three fields in which the significant differences of the low LNCaP cells

or non-metastatic, which had undetectable expression of uPA and uPAR proteins, are represented with asterisks * (P <0.05) (D). FIG. 2 shows that genosuppression of uPA and uPAR expression RNAi in the prostate cancer PC3 cell line. Schematic representation of the sh-uPAuPAR plasmid construct (A). The construct

65 consists of a human CMV promoter and homologous sequences directed against uPA and uPAR. After expression, the strong CMV promoter drives the formation of short hairpin molecules specific for uPA

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and uPAR. The bovine growth hormone (BGH) poly adenylation sequence serves as a signal determining the CMV promoter based on pol II RNA. Dicer / Drosha processes the specific siRNA for uPA and uPAR and the resulting siRNA molecules interact with the uPA and uPAR target genes. This interaction results in the simultaneous genosuppression of the genetic expression of uPA and uPAR. Semi-quantitative reverse-PCR transcription of RNA extracted from PC3 cells transfected with shRNA (B). The mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was co-amplified as a control. Immunoblotting of total protein lysates extracted from PC3 cells transfected with shRNA (C). Both bands of uPA and uPAR are present in cells after decades with simulated, EV and SV. Therefore, each gene-specific RNAsh street shows a significant decrease in the appropriate band. GAPDH was included as a load control. The expression levels of uPA and uPAR proteins were also detected using indirect immunofluorescence in PC3 cells. PC3 cells transfected with the EV, SV and simulated cells were stained positively for immunofluorescence detection of uPA (FITC) and uPAR (Texas Red) (D). Cells transfected with gene-specific rRNA basically changed the cell staining profiles of uPA and uPAR compared to cells transfected with dissimulated EV / SV. Nuclear counterstain

15 obtained with DAPI. (The results are representative of at least three separate experiments). FIG. 3 shows that RNAi genosuppression of the expression of uPA and uPAR inhibits the invasive potential of PC3 cells. The invasive potential of simulated cells and cells transfected with the indicated rRNA plasmids were examined by the Matrigel invasion assay (visual field representative of an experiment) (A). Invasion assays were performed as described herein (see FIG. 1C). The representative number of invasive cells was counted through the matrix with a microscope in three random fields at 200X (B). Each / represents the average DT of the three fields that were counted. The significant difference of the controls (ie, cells simulated or transfected with encoded vector) is indicated with asterisks * (P <0.05). FIG. 4 illustrates that RNAi genosuppression of uPA and uPAR expression inhibits cell proliferation and

25 induces apoptosis in PC3 cells. The viability of PC3 cells transfected with any of the gene-specific plasmid or mRNA controls (cells simulated or transfected with EV / SV) was revealed with the 3- (4,5-dimethylthiazol-2-yl) bromide assay - 2,5-diphenyltetrazolium (MTT) (A). Each bar represents triplicate analysis of mean DT in which the significant difference of the controls is represented by an asterisk * (P <0.05). Representative immunoblotting shows changes in proapoptotic gene expression in genosuppressed PC3 cells for uPA-uPAR (B). GAPDH was used as a load control. Caspase activation was detected in situ with fluorescence labeling (lower panel) using FAM-VAD-FAK, a cell permeable caspase inhibitor that binds to activated caspases (C). Nuclear staining was performed with DAPI (upper panel). A significant number of cells transfected with sh-uPAuPAR have green fluorescence. The bar chart shows quantitative data on the proportion of cells marked with

35 DAPI / FMK-VAD-FAK of three random fields with a confocal microscope (D). The proportion of DAPI to FMK-VAD-FAK increased significantly in cells transfected with sh-uPAuPAR. Significant differences in cells simulated or transfected with EV / SV are indicated with asterisks * (P <0.05). DNA labeling was observed in cells transfected with uPA-uPAR shsh and actinomycin D treated cells (ActD, 0.2 g / ml) (E). An agarose gel was stained with ethidium bromide and photographed with UV light. The DNA markers were electrophoresed as a reference of pairs of kilobases with a band pattern of 2.0, 1.5, 1.0, 0.75 and 0.5 kb (M lane). FIG. 5 demonstrates that RNAi genosuppression of uPA and uPAR expression inhibits downstream signaling in PC3 cells. Immunoblot analysis of total and phosphorylated forms of extracellular signal-regulated kinase (ERK), p38 and JNK in simulated cells transfected with shRNA (A). The

45 PC3 cells transfected with simulated, EV, SV, sh-uPA, sh-uPAR and sh-uPAuPAR were lysed 72 h later and subjected to SDS-PAGE followed by and a transfer with total and phosphorylated forms of ERK, p38 and JNK GAPDH antibodies were used to verify that similar amounts of protein were loaded on each street. Immunoblot analysis of Stat 3 protein in cells simulated and transfected with shRNA (B). Equal amounts of protein were loaded and immunoblotting was performed using phospho-specific Stat 3 antibodies against tyrosine 705 and antibodies against a nonphosphorylated form of Stat 3. GAPDH was included as a loading control. Electrophoretic mobility displacement assay of simulated and transfected cells with shRNA (C). The protein-DNA complexes were separated on a 6% polyacrylamide gel, dried and autoradiographs. Previously, DNA-specific binding activity of nuclear extracts prepared from the transfected cells indicated with shRNA was shown.

55 The position of the free probe is shown. FIG. 6 shows that RNAi genosuppression of uPA-uPAR expression cancels tumor growth in an orthotopic model of mouse prostate tumor. Representative on-site photographs of each treatment group of mice carrying PC3 orthotopic tumors (A). The primary prostate tumor is marked with dashed arrows and solid arrows indicate the position of the metastasis. PC3 cells were transplanted intraprostatically into athymic mice and established PC3 prostate tumors were treated with specific shRNA for uPA, uPAR and uPAuPAR. After 4 weeks of treatment of these constructs, the mice were sacrificed and evaluated for primary prostate tumor growth and visual metastasis. A comparison of dissected prostate tumors from each RNAsh treatment group (B). Each bar represents the average DT of the tumor weight of six animals per group. The significant differences of

65 control groups (ie, simulated or treated with EV / SV) are represented with asterisks * (P <0.05). Protein samples extracted from PC3 prostate tumors of six animals by groups were analyzed using immunoblotting for uPA and uPAR (C) expression levels. GAPDH was included as a load control. FIG. 7 demonstrates that RNAi genosuppression of the expression of uPA and uPAR simultaneously cancels tumor growth in an orthotopic model of mouse prostate tumor. On-site photographs of each group

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5 of treatment of mice bearing PC3 orthotopic tumors (A). The primary prostate tumor is marked with dashed arrows and solid arrows indicate the position of the metastasis. PC3 cells were transplanted intraprostatically into athymic mice and established PC3 prostate tumors were co-injected with both sh-upA and sh-uPAR vectors. After 4 weeks of treatment of these constructs, the mice were sacrificed and evaluated for primary prostate tumor growth and visual metastasis. A comparison of dissected prostate tumors from each RNAsh treatment group (B). Each bar represents the average DT of the tumor weight of six animals per group. Significant differences of control groups (ie, simulated or treated with EV / SV) are represented by asterisks * (P <0.05). Protein samples extracted from PC3 prostate tumors of six animals by groups were analyzed

15 using immunoblot for expression levels of uPA and uPAR (C). GAPDH was included as a load control. Representative sections of hematoxylin and eosin of PC3 mouse mouse prostate orthotopic tumors (D). Primary prostate tumors were collected from each treatment group at the end of the experiment. The tumors were fixed in formalin and embedded in paraffin. Tissue sections (5 m) were prepared and stained with H and E for histopathological analysis. FragEL DNA staining of microdissected paraffin sections of prostate tumors established from the indicated treatment groups (E). End labeling assays of the DNA fragment were performed. The results are shown with a 40X increase except for the box, which has a 200X increase. Bar chart showing quantitative data of cells labeled with fragEL DNA from six random fields per treatment group (F). Significant differences in the control groups are indicated with asterisks * (P <0.05).

25 FIG. 8 shows a schematic representation of the representation of siRNA for uPAR and MUMP-9 of the pUM vector. Plasmid pcDNA 3 constructs were developed with two complementary inverted repeats directed by a CMV promoter directed against uPAR and MMPs-9. The CMV promoter directs the formation of a double hairpin structure which, in turn, is processed by double-stranded RNA that recognizes the DICER enzyme to form viable siRNA molecules. The stability of the double hairpin molecule is ensured because the secondary structure of the molecule reminiscent of an mRNA molecule that has a poly A tail directed by a poly-a bovine growth hormone (BGH) signal sequence. FIG. 9 illustrates whether long hairpin RNA (hp) is processed to siRNA; if molecules are transfected into SNB19 cells with control / EV, SV, puPAR, pMMP-9 and pUM; if the cells were also transfected with an unrelated construct directed to GFP cells in cells other than GFP to determine the processing of the molecules

35 appropriate siRNA. Small RNA molecules fractionated on a 2% agarose gel were allowed to hybridize with sense oligo labeled with appropriate DIG in the presence of 6x SSC. The resulting hybrid solution was made on a 15% polyacrylamide gel and electrotransformed on a nylon membrane. The membrane was processed to visualize the DNA hybrid: 21 bp RNA according to the manufacturer's instructions. The probes used are represented as numbers (see Table 1), 1-suPAR, 2-sMMP-9 and 3-sGFP (FIG. 9A). SNB19 cells were transfected with control / EV (lane a), SV (lane b), puPAR (lane c), pMMP-9 (lane d) and pUM (lane e) according to conventional protocols known to those skilled in the art. matter. 72 h later, the total RNA was isolated and the first strand of cDNA was synthesized using a cDNA synthesis kit (Invitrogen) (FIG. 9B). The PCR reaction was prepared using the first strand of cDNA as the template for uPAR and Mump-9; PCR for GAPDH was also prepared to serve as a load control

45 (see Table 1). FIG. 10 characterizes Western blot analysis for uPAR and gelatin zymography for MMP-9. SNB19 cells were transfected with simulated, an empty / encoded vector and a vector encoding individual or bicistronic siRNA for uPAR and Mump-9 (puPAR, pMMP-9, pUM). (A) Western blot analysis of uPAR protein expression in cell lysates of SNB19 cells transfected with EV / SV, puPAR, pMMP9 and pUM. Western blot analysis was performed using an antibody specific for uPAR. GAPDH was flooded detected simultaneously to verify the loading of similar amounts of cell lysates. (B) Conditioned media containing equal amounts of protein (20 µg) of transfected cells of EV / SV, puPAR, pMMP-9 and pUM were mixed with Laemmli sample buffer and made in 10% SDS-PAGE gels that contained 0.1% gelatin to determine the activity of MMP-9 with

55 jelly zymography. FIG. 11 demonstrates that negative regulation mediated with uPAR siRNA and MUMP-9 reduces proliferation of glioma SNB19 cells. In summary, 5 x 104 SNB19 cells transfected with EV / SV, puPAR, pMMP-9 and pCM were seeded in 96-well microplates coated with VN under serum-free conditions. The number of viable cells was evaluated with MTT assay. FIG. 12 illustrates that RNAi lower levels of SPAR and MMP-9 as shown with immunohistochemical analysis with and tumor-induced angiogenesis. (A) SNB19 cells were transfected with EV, SV, puPAR, pMMP-9 and pUM. Control or untransfected cells were also used. 72 h after transfection, the cells were mixed and processed to visualize the expression of uPAR and MMP-9 in vivo. Cells were mounted using DAPI mounting media to visualize the nucleus. (B) SNB19 cells (2 x 104) are

65 were seeded on 8-well chamber slides and transfected with simulated EV, puPAR, pMMP-9 and pUM. After 24 h of incubation, the medium was removed and the cells were cocultured with 4 x 104 cells of the human microvascular dermal endothelium. After 72 h, the endothelial cells were investigated with antibody to factor VIII antigen or stained with H and E and examined with a confocal scanning laser microscope. (C) Quantification of angiogenesis in cultures transfected with simulated EV, puPAR, pMMP-9 and pUM vector or pUM vector. Values are the mean ± D.T. of three different experiments. Angiogenesis Inhibition

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5 tumor in pUM vector with mouse dorsal window test (D). PV, previously existing vasculature, TN, tumor induces vasculature. FIG. 13 demonstrates that siRNA for uPAR and MMP-9 inhibits the invasion of SNB 19 cells. SNB19 (1 x 106) cells transfected with EV / SV, puPAR, pMMP-9 and pUM were allowed to migrate through Transwell inserts coated with Matrigel (pores of 8 µm) for 24 h. Cells that invaded through Matrigel-coated inserts were stained, counted and photographed with light microscopy with a 20X magnification. (A) The invasion percentage was quantified. Values are the mean ± D.T. from five different experiments (P <0.001). SNB19 cell spheroids (fluorescence) 100-200 µm in diameter were selected, transfected with EV / SV, puPAR, puMMP-9 and pUM and co-cultured with fetal rat brain aggregates (green fluorescence). The progressive destruction of fetal rat brain aggregates and the

Invasion of SNB19 cells was observed for 72 h using confocal laser scanning microscopy. (C). The remaining volumes of brain aggregates or tumor spheroids at 24, 48 and 72 h were quantified using image analysis software (D). FIG. 14 analyzes the RNAi-mediated regression of intracerebral aggregates previously established in tumor growth. Glioblastoma SNB19-GFP cells were injected intracerebrally (2 x 10 6 cells in 10 µl phosphate buffered saline) in athymic mice. After 10 days, simulated EV, puPAR, pMMP-9 and pUM were used (150 µg of each vector were injected into the brain using Alzet mini pumps at the rate of 0.25 µl / h (eight mice in each group) Sections of tumor photomicrographed for fluorescence with GFP (A) were examined and subsequently stained with hematoxylin and eosin (B) Semi-quantification of tumor volume in groups treated with simulated EV, puPAR, pMMP-9 and pUM vector

25 after 4-6 weeks after intracranial injection of these cells (C). The data shown are values of ± D.T. of eight animals from each group. In another set of experiments, 10 days after intracerebral injection of SNB19 GFP cells, the pUM vector was injected intraperitoneally twice at an interval of 3 days and the animals were sacrificed after 4 months. FIG. 15 shows that RNAi-mediated negative regulation of uPAR and MMP-9 reduces phosphorylation of ERK, MAPK and AKT. Western blot analysis of total and phosphorylated forms of MAPK, ERK and AKT. SNB19 cells were transfected with EV / SV, puPAR, MMP-9 and pUM on VN coated plates and serum-free conditioned cells were lysed 72 h later and subjected to SDS-PAGE and immunoblotted with total and phosphorylated antibody forms MAPK, ERK and AKT. GADPH antibodies were used to verify that similar amounts of protein were loaded on each street.

35 FIG. 16 is a schematic presentation of uPAR and MMP-9 vector (A) and cellular events on the cell surface (B). After plasminogen activation in plasmin, which in turn activates MMPs, release of uPA from various growth factors after ECM degradation. The schematic presentation demonstrated the implication of integrins in several signaling pathway molecules. FIG. 17 is a schematic representation showing the formation of hRNA molecules from a single double-directed inverted CMV repeat construct for cathepsin B and uPAR. The CMV viral promoter directs the formation of an RNA molecule that has autocomplementary inverted repeats for cathepsin B and uPAR. FIG. 18 shows Western blot analysis for uPAR and cathepsin B. SNB19 cells were transfected with simulated, an empty vector and a vector encoding siRNA for cathepsin B and uPAR (pCU).

Western blot analysis of cathepsin B (A) uPAR (C) protein lysates of SNB19 cells transfected with simulated, empty vector and pCU was performed using a specific antibody for cathepsin B and uPAR. The β-actin was subjected to immunoinvestigation simultaneously for load control. Quantification of cathepsin B (B) and uPAR protein (D) was obtained by scanning the autoradiograms with densitometry. FIG. 19 shows that RNAi inhibits the formation of capillary networks induced by tumor cells. SNB19 cells were transfected with simulated, empty vector, pC, pU and pCU for 24 hours. Next, the cells co-cultured with human dermis endothelial cells for 48 h. After incubation, the cells were fixed and blocked with 2% bovine serum albumin for 1 h and the endothelial cells were investigated with antibody for factor VIII antigen. (Factor VIII Antibody, DAKO Corporation,

55 Carpinteria, California) or staining with H and E and examined with a confocal laser scanning microscope (A). Quantification of angiogenesis in cocultures infected with simulated, empty vector or pCU vector (B). Inhibition of tumor angiogenesis in SNB19 cells infected with pCU vector with mouse dorsal window assay (C). PV, previously existing vasculature, TN, tumor induced vasculature. FIG. 20 demonstrates that RNAi inhibits the migration and invasion of glioma cells. The spheroids of SNB19 GFP were infected with simulated, empty vector, pC, pU and pCU. After 72 h, single glioma spheroids were placed in the center of a vitronectin-coated well of a 96-well plate and cultured for 48 h. At the end of the migration trial, the spheroids were fixed and photographed (A). The migration of spheroid cells was measured using a microscope calibrated with a platform and an ocular micrometer (B). The data shown are the mean value ± D.T. of the results of four experiments

65 independent of each group. SNB19 cells were trypsinized 72 h after transfection with simulated, empty vector, pC, pU and pcU, washed with PBS and resuspended in serum free medium. The essays of

5

fifteen

25

35

Four. Five

55

65

Invasion was performed in a 12-well Transwell unit (Costar, Cambridge, Massachusetts) in polycarbonate filters with 8 µm pores coated with Matrigel. After an incubation period of 24 h, the cells that had passed through the filter in the lower corridors were stained, became frequent and photographed with light microscopy (C). The invasion percentage was quantified (D). Values are the mean ±

D.T. of 5 different experiments (P <0.001). The spheroids of SNB19 cells were transfected with simulated, empty vector, pC, pU and pcU and stained with DiI and fetal rat aggregates stained with DiO were co-cultured. The progressive destruction of fetal brain aggregates with tumor spheroids (E) was observed. Quantification of the remaining fetal brain aggregates with SNB19 spheroids infected with empty vector, pC, pU or pCU vector (F). FIG. 21 shows that the negative regulation mediated by uPAR RNAi and Cathepsin B reduces proliferation of glioma SNB 19 cells. Proliferation test In summary, 5 x 104 SNB19 cells transfected with PBS, EV, SV, pU, pC and pCU were seeded in 96-well microplates coated with VN under serum-free conditions. The number of viable cells was evaluated with the MTT assay. The mean values (± D.T.) of three experiments are shown separately. FIG. 22 illustrates that the negative regulation mediated by uPAR siRNA and Cathepsin B reduces phosphorylation of ERK and FAK. Western blot analysis of ERK, FAK, total and phosphorylated proteins using their specific antibodies after transfection of SNB19 cells with simulated constructs, EV, SV, pU, pC and pCU. Β-actin levels served as load control. FIG. 23 shows the RNAi-mediated regression of previously established tumor growth. SNB19 GFP tumor cells were injected intracerebrally with the help of a stereotactic framework in athymic mice. After 1 week, either of an empty vector or a vector that expresses siRNA for cathepsin B and uPAR (pCU) or separately with pC or pU was injected into the brain using a mini osmotic pump from Alzet. The photomicrographed tumor sections were observed for GFP fluorescence (A) and subsequently stained with hematoxylin and eosin (B). Semi-quantification of tumor volume in groups treated with simulated / empty vector, pU, pC and pCU vector after 5 weeks. The data shown are the values of ± D.T. of 6 animals from each group (* P <0.001) (C). FIG. 24 is a schematic representation of the formation of hRNA molecules from a single tri-inverted repeat constructor, directed by CMV for uPAR, uPA and MMP-9. The potent CMV viral promoter directs the formation of a rRNA molecule possesses inverted autocomplementary repeats for uPA, uPAR and MMP-9. FIG. 25 is a Western blot analysis, fibrin / gelatin zymography and immunohistochemistry of uPA and MMP-9. SNB19 cells were transfected with simulated or transfected with an empty vector / encoded vector (EV / SV) and vectors encoding siRNA uPAR (puPAR), uPA (puPA), MMP-9 (pMMP-9) and a combination of three points (pU2M). After an incubation period of 3 days, the total cell lysates were prepared in extraction buffer and 50 µg of protein from these samples were separated with 12% non-reducing SDS-PAGE and immunoblotted with anti-uPAR antibody (A) . The GAPDH underwent immunoinvestigation simultaneously with a load control. The conditioned medium was collected from these samples (20 µg) and gelatin and fibrin zymography was performed to detect the activity of MMP-9 (B) and uPA (C). (D) SNB19 cells (1 x 104) were seeded on Lab-Tek II camera slides and transfected with simulated or transfected with EV / SV and vectors encoding siRNA puPA, puPAR and pMMP-9 either individually or together (pU2M). After 72 hours, the cells were fixed, washed for 1 hour with blocking buffer and stained for expression of uPAR, uPA and MMP-9 using antibodies specific for uPA, uPAR and MMP-9. FIG. 26 illustrates the inhibition of glioma angiogenesis and invasion with siRNA constructs. SNB19 cells (2 x 104) were seeded on 8-well chamber slides and transfected with EV / SV directors encoding siRNA uPAR (puPAR), uPA (puPA), MMPs-9 (pMMP-9) and a combination of the three points (pU2ND. After a 24-hour incubation period, in the middle it was removed, the cells were cocultured with either 4 x 104 human endothelial cells or 4 x 104 endothelial cells alone and cultured in the presence of conditioned media. After 72 hours the endothelial cells were stained for factor VIII antigen in the cocultures (green fluorescence) The cells grown in the preserved conditioned media were stained with H and E and examined with either a fluorescent microscope or a microscope bright field (A). (B) Quantification of angiogenesis in cocultures infected with EV / SV, puPAR, puPA, pMMP-9 and pU2M vectors. Values are the mean ± SD of four experiments. SNB19 cells are Trypsined 72 hours after transfection with EV / SV, puPAR, puPA, pMMP-9 and pU2M, washed with PBS and re-suspended the medium without serum. (C) invasion assays were carried out in a 12-well Transwell unit in polycarbonate filters with 8 µm pores coated with matrigel (0.7 mg ml-1). After an incubation period of 24 hours, the cells that had passed through the filter in the lower wells were stained, the bright field microscope was photographed. (D) the invasion percentage was quantified. FIG. 27 shows the inhibition and regression of tumor invasion and growth capacity with siRNA with spheroid and intracranial assays. (A) The ability to invade glioma spheroids is by co-cultivation of glioma spheroids with aggregates from the fetal rat brain. SNB19 cell spheroids were transfected with EV / SV, puPA, puPAR, pMMP-9 and pU2M and stained with DiI and co-cultured with fetal rat brain aggregates stained with DiO. Sections 1 µm thick in series were obtained from the surface through the center of the cocultures with a confocal laser scanning microscope at the indicated time points. (B) The remaining volume of rat brain aggregate transfected with EV / SV, puPA, puPAR, pMMP-9 and pU2M was measured. Values are the mean ± SD of three experiments. (C, D) RNA-mediated regression of previously established tumor growth. SNB19 GFP cells in suspension (2 x 106 in 10 µl of serum-free medium) were injected intracranially. One week later, the mice were injected with any of the EV / SV or siRNA expression vectors (puPAR, puPA, pMMP-9 and pU2M) using a

image7

5 Alzet mini osmotic pump (constructs diluted to 1.5 µg ml-1 in PBS and injected at 0.25 µg hour-1, with six mice in each group). After a 5-week follow-up period, the mice sacrificed, their brains were extracted, embedded in paraffin and sectioned. Sections were observed with fluorescence microscopy for cells expressing GFP. (D) The semiquantification of tumor volume in groups treated with control, EV / SV, puPAR, puPA, pMMP-9 and pU2M was evaluated after 5 weeks. Data are shown as mean ± SD of six animals of each group. FIG. 28 demonstrates that negative regulation mediated with uPAR RNAi, uPA and MMP-9 reduces ERK phosphorylation. Western blot analysis of total and phosphorylated ERK protein (pERK) after transfection of glioblastoma cells with constructs of EV / SV, puPAR, puPA, pMMP-9 and pU2M. GAPDH levels served as load control.

15 FIG. 29 is a schematic representation of siRNA expression for cathepsin B and MMP-9 of pCM vector constructs: pCDNA 3 plasmid developed with two complementary inverted repeats directed by a CMV promoter directed against cathepsin B and MMP-9. The CMV promoter directs the formation of a double hairpin structure that, in turn, was processed with double-stranded RNA that recognizes the DICER enzyme to form viable siRNA molecules. The stability of the double hairpin molecule was ensured due to the secondary structure of the molecule reminiscent of an mRNA molecule that has a poly A tail directed by a bovine growth hormone (BGH) poly-a signal sequence. FIG. 30 confirms that interference RNA decreased cathepsin B and MMP-9 levels in SNB19 cells. Total cell lysates and serum-free medium were collected from SNB19 cells transfected with simulated, empty vector or a vector encoding siRNA for MMP-9 (pM) and cathepsin B (pC) and together (pCM).

Subsequently, 30 µg of protein was separated from these samples under non-reducing conditions in 8% to 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were investigated with antibodies to Cathepsin B (A) and MMP-9 (C) and with appropriate secondary antibody (horseradish peroxidase conjugate) and developed according to the manufacturer's protocol (Amersham, Arlington Heights, Illinois) . The β-actin was immunodetected simultaneously to verify the loading of similar amounts of cell lysates. The MMP-9 activity of SNB19 cells infected with empty vector, pC, pM or pCM vector for 3 days in serum-free medium and was determined with gelatin zymography (B). FIG. 31 shows that RNAi inhibits the formation of capillary networks induced by tumor tumor cells. SNB19 cells were transfected with simulated, empty vector, pM, pC and pCM for 24 h. Next, the cells were cocultured with human dermis endothelial cells for 48 h. After incubation, the

35 cells were fixed and blocked with 2% bovine serum albumin for 1 h and the endothelial cells were investigated with factor VIII antigen antibody. (Factor VIII Antibody, DAKO Corporation, Carpinteria, California) and were examined with fluorescence microscopy after researching with a secondary antibody conjugated with appropriate FITC. Endothelial cells were grown in the presence of SNB19 conditioned media (control, empty vector, PM, pC, or transfected pCM) that were stained with H and E and photographed (A). Quantification of angiogenesis in cocultures infected with simulated, empty vector or pCM vector (B). Inhibition of tumor angiogenesis in SNB19 cells infected with pCM vector by mouse dorsal cutaneous fold assays (C). Pre-existing PV vasculature, TN tumor induced vasculature. Photographs were taken with light microscopy (upper panel) and for fluorescence with FITC (lower panel) to determine the presence of newly developed vasculature.

45 FIG. 32 demonstrates that RNAi inhibits the migration and invasion of glioma cells. SNB19 GFP spheroids were infected with simulated, empty vector and a vector encoding siRNA for cathepsin B and MMP-9 (pCM). After 3 days, single glioma spheroids were placed in the center of a 96-well plate coated with the vitronectin-coated wells and cultured for 48 h. At the end of the migration trial, the spheroids were fixed and photographed (A). SNB19 cells were trypsinized 3 days after transfection with simulated, empty vector and a vector encoding siRNA for cathepsin B and MMP-9 (pCM), washed with PBS and resuspended in serum free medium. Invasion assays were performed in a 12-well Transwell unit (Costar, Cambridge, Massachusetts) on polycarbonate filters with 8 µm pores coated with Matrigel. After an incubation period of 24 h, the cells that had passed through the filter in the lower wells were stained, counted and photographed with light microscopy (B). The

55 spheroids of SNB 19 cells were transfected with simulated, empty vector and a vector encoding siRNA for cathepsin B and MMP-9 (pCM) and stained with DiI and co-cultured with fetal rat aggregates stained with DiO. Progressive destruction of fetal brain aggregates was observed with tumor spheroids (C). FIG. 33 shows the RNAi-mediated regression of previously established tumor growth. SNB19 GFP tumor cells were injected intracerebrally with the help of a stereotactic framework in athymic mice. After 1 week, either an empty vector or a vector expressing siRNA for cathepsin B and MMP-9 (pCM), cathepsin B (pC) or MMP-9 (pM) was injected into the brain using a mini osmotic pump Alzet Photographs of tumor sections were observed with GFP fluorescence (A) and subsequently stained with hematoxylin and eosin (B). Semiquantification of tumor volume was performed in groups treated with simulated vector / vacuum, pM vector, pC and pCM after 5 weeks. The data shown are the values of ±

65 D.T. of 6 animals from each group (* P <0.001) (C). In another experiment, 10 days after intracerebral injection, the pCM vector was injected intraperitoneally twice and the animals were then sacrificed.

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4 months (D). FIG. 34 is a schematic representation of pol II RNA promoter (CMV) for the induction of RNAi. FIG. 35 is a schematic representation of pol III RNA (U6) based promoter for the induction of RNAi.

5 FIG. 36 is a schematic representation of recombinant adeno virus production. FIG. 37 indicates SNB19 GFP cells transfected with RNAi for expression of GRP or GRP showing simulated. FIG. 38 shows the expression of OAS in SNB 19 cells transfected with RNAi vectors directed by pol II RNA (CMV) or pol III RNA (U6) that was determined with RT-PCR. RT-PCRT for GAPDH served as a control. FIG. 39 demonstrates the negative regulation of uPAR and uPA as determined by Western blot analysis and fibrin zymography of lysates of SNB 19 cells of affected cells with either promoters directed by U6 or CMV. RNAi plasmids for encoded vector (SV), plasmid expressing RNAi for uPAR (puPAR), uPA (puPA) and bicistronic construct of uPAR-uPA (pU2). GAPDH is

15 investigated for load control. FIG. 40 is a schematic representation of siRNA expression constructs used to determine the induction of cellular immune response. FIG. 41 shows the determination of the expression of 2'5'-oligoadenylate synthetase (OAS1) in SNB19 cells transfected with circular (C), linear (L) or poly A expression cassette with suppressed signal (∆A) for empty vector ( EV), encoded vector (SV), siRNA expression constructs for uPAR (puPAR), uPA (puPA) and bicistronic construct for uPAR and uPA (pU2) with RT-PCR. RT-PCR was normalized with GAPDH. The expression of OAS1 was quantified as shown. FIG. 42 is a schematic representation of the predicted secondary structure of EV and SV transcripts that do not show RNA-like RNA-inducible type structure and predicted transcript of puPAR, puPA and pU2

25 with poly A and without poly A pU2∆A). FIG. 43 is a schematic representation of the partial secondary transcript structure of pU2 showing secondary structure in space. The secondary structure in space does not resemble that of mRNA. FIG. 44 shows RT-PCR in a total RNA isolated from control (C), antisense uPAR (such as uPAR), antisense uPA (such as uPA) and RNAi constructs for uPAR (puPAR), uPA (puPA), uPAR-uPA ( pU2), GFP (pGFP), empty vector (pEV), and encoded vector (pSV) for RNA levels in uPA, uPAR and OAS. GAPDH served as a control. FIG. 45 our hybridization in situ of the CMV promoter in mice injected IP. Probe was used in skull sections of mice infected intraperitoneally with saline solution (Simulated), empty vector

35 (EV), encoded vector (SV), RNAi expression vectors for uPAR (puPAR), uPA (puPA), and bicistronic construct of uPAR-uPA (pU2), for the presence of CMV promoter containing DNA by labeling with DNA probe with alkaline phosphatase (AP). AP activity was detected with Western Blue substrate for AP (Promega, Madison, WI).

Detailed description

Small hairpin RNAs (rRNAs), also called small interference RNAs (siRNAs), human target genes such as uPA and uPAR to inhibit tumor growth, tumor invasion, and tumor proliferation. The siRNA constructs significantly inhibited the expression of uPA and uPAR at both mRNA and protein levels in a highly metastatic prostate cancer PC3 cell line. The genosuppression of uPA-uPAR in PC3 cells resulted in a significant reduction of tumor cell invasion as indicated, for example, with a Matrigel invasion assay. Simultaneous silencing of the genes for uPA and uPAR using a single plasmid constructor that expresses the shRNAs for both uPA and uPAR significantly reduced cell viability and also resulted in the induction of apoptopic cell death. The siRNA for uPA and uPAR also nullified the signaling of uPA-uPAR to downstream target molecules such as 1/2 kinases (ERK1 / 2) regulated by extracellular signal and signal transducer and transcription activator 3 (Stat 3). Intratumoural injection with a plasmid construct that expresses the shRNAs for uPA and uPAR significantly inhibited established tumor growth and survival in an orthotopic model of mouse prostate cancer. Evidence of a signaling network that runs downstream of uPA-uPAR to advance

55 actively invading tumor cells, proliferation and survival of prostate cancer cells is uncovered. The uPA and uPAR targeting directed by RNAi and the corresponding siRNAs are new therapeutic agents for cancer therapy, which includes prostate cancers.

Degradation of uPAR and MMPs-9 target RNA mediated by siRNA in human glioma cell lines resulted in tumor inhibition. The siRNA directed towards uPAR and MMP-9 achieved a specific inhibition of uPAR and MMPs-9. A bicistronic construct (PUM) inhibited the formation of capillary structures in angiogenesis models both in vitro and in vivo. Blocking the expression of uPAR and MMP-9 resulted in significant inhibition of glioma tumor invasion in Matrigel and spheroid invasion assay models. The siRNA for uPAR and MMP-9 inhibited cell proliferation and reduced the levels of the phosphorylated forms of 65 molecules of the MAPK, ERK and AKT signaling pathway when compared with precursor SNB19 cells and transfected with empty vector / encoded vector ( EV / SV). In addition, the use of RNAi to direct simultaneously

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two protease molecules and injecting these constructs intracerebrally in vivo using Alzet mini pumps or intraperitoneal injections resulted in significant regression of previously established intracerebral tumor growth. The use of fork siRNA expression vectors for uPAR and MMP-9 provides an effective therapeutic tool for cancer therapy, including glioblastoma.

5 In another embodiment, the RNAi approach silenced the expression of uPAR and cathepsin B. The RNAi is to inhibit the expression of proteases involved in the degradation of the extracellular matrix, a characteristic element of tumor progression. UPAR and cathepsin B RNAi reduced glioma cell invasion and angiogenesis in in vitro and in vivo models. Intratummoral injections of plasmid vectors expressing hRNA (siRNA) for uPAR and cathepsin B resulted in the regression of previously established intracranial tumors. The siRNA for uPAR and cathepsin B inhibited cell proliferation and reduced pERK and pFAK levels compared to controls. RNAi works in human glioma cells and provides a basis for genetic therapy for cancer, which includes glioblastoma.

15 The RNAi approach silences the expression of uPA, uPAR and MMP-9 in tumor cells. A DNA template directed by the cytomegalovirus (CMV) promoter in a single trichystronic construct induced RNAi triggered by hairpin RNA (hRNA) to inhibit the genetic expression of uPA, uPAR and MMP-9 with a single construct. The levels of uPAR protein and the enzymatic activity of uPA and MMP-9 were found to decrease significantly in cells transfected with a plasmid that expresses hairpin siRNA for uPAR, uPA and MMP-9. SNB19 cells transfected with pU2M significantly decreased the expression of uPA, uPAR and MMP-9 compared to cells with simulated and transfected with EV / SV, determined with immunohistochemical analysis. Individual constructs for these molecules resulted in a specific inhibition of their respective protein levels, as demonstrated by immunohistochemical analysis. After transfection with a plasmid vector expressing RNAs for uPA, uPAR and MMP-9, glioma cell invasion was delayed in

25 comparison with simulated and EV / SV treated groups, demonstrated with a Matrigel invasion assay and spheroid invasion assay. Negative regulation of uPA, uPAR and MMP-9 using RNAi inhibited angiogenesis in an in vitro model (coculture). Direct intratumoral injections of plasmid DNA expressing hRNA for uPA, uPAR and MMP-9 also significantly reversed previously established intracranial tumors in nude mice. RNAi-treated cells for uPAR, uPA and MMP-9 showed acid levels of pERK compared to precursor SNB 19 cells and treated with EV / SV. Simultaneous repression of uPAR, uPA and MMP-9 is a therapeutic tool to treat cancers.

A single-stranded DNA hairpin RNA directed by cytomegalovirus (CMV) (mRNA, siRNA) promoter blocked the genetic expression of MMP-9 and cathepsin B. Transfection of a plasmid vector that

35 expresses RNAds for MMP-9 and cathepsin B significantly inhibited the expression of MMP-9 and cathepsin B and reduced the invasive behavior of the glioblastoma cell line, SNB 19, in Matrigel and spheroid invasion models. Negative regulation of MMP-9 and cathepsin B using RNAi in SNB 19 cells also reduced the cell-cell interaction of human microvascular endothelial cells, resulting in the alteration of capillary network formation in both in vitro and in vitro models. alive. Direct intratumoral injections of plasmid DNA expressing hRNA for MMP-9 and cathepsin B significantly inhibited the growth of glioma tumor established in invasion in intracranial tumors in vivo. Intraperitoneal (ip) injections of plasmid DNA expressing hRNA for MMP-9 and cathepsin B completely reversed previously established tumors for a significant period of time. Simultaneous mRNA-mediated management of MMP-9 and cathepsin B is an appropriate treatment methodology for human gliomas.

45 The CMV promoter-directed mRNA, based on plasmid that targets uPAR, uPA and MMP-9, either individually or simultaneously, induces RNAi in the human glioma cell line, SNB 19. Negative mediated regulation by RNAi, simultaneous with uPAR, uPA and MMP-9 in human glioma cells, SNB 19, caused:

(one)

Inhibition of invasion and angiogenesis in vitro.

(2)

Regression of intracranial tumors previously established in nude mice in vivo.

(3)

Reduction of phosphorylation of ERK signaling molecules 1 and 2.

55 siRNAs or siRNAs or mRNAs directed from a circular plasmid (eg, uPA, uPAR, MMP-9, and cathepsin B

or any combination thereof) are suitable for inducing RNA interference in vitro. Circular plasmid siRNAs are stable and do not induce an undesirable immune response, as demonstrated by induction with OAS1. Some linear constructs of uPA, uPAR, MMPs-9, and cathepsin B or any combination thereof are also suitable for the induction of RNAi.

The siRNAs or siRNAs or hRNAs disclosed herein include nucleic births that basically consist of autocomplementary sequences of uPA, uPAR, MMP-9, cathepsin B or a combination thereof. The loop region and the spacer (intervention pressures) can vary in both length and length.

65 sequence depending on the target sequence, the construct, and therapeutic use. The nucleic acid molecules expected herein may be appropriately modified with nucleic acid analogs, derivatives, or any suitable modification to increase the stability or efficacy of the induction of RNAi. The nucleic acid molecules disclosed herein may also be administered in combination with other tumor specific immune activating agents, tumor targeting agents, and any suitable pharmaceutically acceptable carrier or whistle. The nucleic acid molecules that are

image10

5 disclosed herein may also be administered in conjunction with other cancer therapies such as radiation therapy or chemotherapy.

Examples

Examples that are not covered by the claims are for illustrative purposes only.

Example 1. The expression of endogenous uPA and uPAR protein is associated with the ability of in vitro invasion of human prostate cancer cells.

15 PC3 cells are highly metastatic, while DU145 and LNCaP cells are moderately and poorly metastatic, respectively. uPA and its uPAR receptor are involved in tumor invasion and metastasis. The levels of these proteins in the three human prostate cancer cell lines with different metastatic potentials were compared. As shown in FIG. 1A, the levels of uPA and uPAR proteins were significantly higher in PC3 and DU145 cells compared to sparsely metastatic LNCaP cells, which expressed undetectable levels of these proteins. A similar trend was observed in the activity of uPA as evaluated by fibrin zymography (FIG. 1B). Therefore, uPA uPAR protein levels as well as uPA activity were positively correlated with their known metastatic potentials. The ability of prostate cancer cells to invade Matrigel, a gel layer formed by a membrane protein base, was examined. This trial is a well established in vitro model to assess the

25 capacity for tumor invasion. The highly metastatic prostate cancer PC3 cell line showed the highest levels of invasion capacity followed by the DU145 and LNCaP cell lines in an order consistent with their known metastatic potentials (FIG. 1C and 1D). PC3 cells were 14 times more invasive and DU145 were 9 times more and more massive than LNCaP cells. A strong correlation between the levels of uPA and uPAR proteins and the invasive capacity of human prostate cancer cells with different metastatic potentials was demonstrated. PC3 di DU145 cell lines were more and more massive than LNCaP cells, consistent with their known metastatic potentials. There is a strong correlation between the expression patterns of uPA and uPAR and the invasive potential of the prostate cancer cell lines used (FIG. 1). Increased expression of uPA and uPAR in prostate cancer cell lines is associated with increased invasion capacity and metastatic potential.

Example 2. Effective genosuppression of the genetic expression of uPA and uPAR in PC3 cells of human prostate cancer using RNAi.

The biological role of uPA and uPAR in the evolution of the prostate tumor was investigated using small hairpin RNA to genosuppress the genetic expression of endogenous uPA and uPAR in the human prostate cancer PC3 cell line, which expresses uPA and uPAR as well as a high metastatic potential. PcDNA3-CMV vectors were developed containing small hairpin constructs capable of generating 19 or 21 nt duplex RNAi oligonucleotides corresponding to either uPA or uPAR. In addition, a single bicistronic construct directed by a cytomegalovirus promoter (CMV) was constructed to deliver small double directed forks

45 against uPA as well as uPAR to test the calcium and inhibition expression simultaneously of two endogenous genes (FIG. 2A). Vectors that express the siRNAs for uPA, uPAR and the uPAuPAR combination were transfected into PC3 cells.

As shown in FIG. 2B, analysis of cells transfected with mRNA for the expression of uPA and uPAR by semiquantitative reverse transcription PCR demonstrated a specific reduction in mRNA levels for each gene with respect to cells transfected with EV / SV or simulated cells. However, the effect of the RNAi was greater with the siRNA vector that simultaneously targets uPA and uPAR (FIG. 2B). The analysis of no transfer of cell extracts was performed to determine if the decrease in mRNA expression, as observed, correlated with the decrease in the translation of the genetic product. A

A similar trend was also observed with the worthy transfer assay (FIG. 2C). No effects of RNAi were observed in GAPDH expression, which was used as an internal control for specificity and carp at the mRNA level as well as at the protein level. In addition, cells transfected with EV / SV also showed that uPA and uPAR genosuppression directed by RNAi is specific (FIG. 2B and 2C).

In addition, the effects of gene-specific rRNAs on the expression of uPA and uPAR proteins were detected in PC3 cells using double immunostaining with anti-uPA and anti-uPAR antibodies. As shown in FIG. 2D, the staining of uPA and uPAR was radically reduced with gene-specific rRNAs compared to cells transfected with EV / SV. Double immunostaining of uPA and uPAR decreased completely in cells transfected with sh-uPAuPAR (FIG. 2D). In contrast, PC3 cells transfected with EV and SV had an intensity of

65 staining and patterns similar to simulated cells. These immunofluorescence studies were confirmed with RT-PCR and immunoblot analysis.

Simultaneous inhibition of two genes using a plasmid-based siRNA system is a useful tool. The RNAi effectively regulates the mRNA of uPA and uPAR as well as the expression of proteins in the prostate cancer PC3 cell line (FIG. 2B and 2C). These gene-specific RNAi plasmids reduced the extent of uPA and uPAR substantially compared to cells simulated or transfected with EV / SV.

5 Immunohistochemical staining data showed that cells simulated or transfected with EV / SV rebel positive staining for uPA and uPAR, while cells transfected with sh-uPAuPAR were hardly stained, with the exception of nuclear staining with DAPI, which suggests the genosuppression of the expression of the uPA and uPAR proteins (FIG. 2D). Similarly, the intensity of immunostaining for uPA and uPAR was reduced with the gene specific RNAi.

Example 3. The genosuppression of uPA and uPAR with RNA inhibited the invasion in Matrigel of PC3 cells.

One of the functions of uPA and uPAR is the promotion of invasion, a necessary process for tumor metastasis. The impact of the genosuppression of uPA and uPAR on invasion of PC3 cells was evaluated with a Matrigel invasion assay using cells transfected with shRNA. When compared to simulated cells or cells transfected with EV / SV, cells transfected with sh-uPAuPAR exhibited a substantial reduction in invasion capacity (FIG. 3A). The invasion of PC3 cells was reduced to 75% of that of the controls (ie, cells simulated or transfected with EV / SV) with sh-uPA and 90% with sh-uPAuPAR (FIG. 3B). Although the genosuppression of uPAR alone did not show a significant inhibition of invasion, the genosuppression of uPA as well as of uPAR had a significant effect (FIG. 3A and 3B), suggesting that the invasion of PC3 cells in Matrigel is basically regulated by the coordinated function of uPA and uPAR. These results show that the expression of uPA and uPAR is necessary for the invasion of prostate cancer as well as metastasis. The effect of sh-uPAuPAR was so significant that cells could barely invade through the Matrigel membrane, suggesting that RNAi had significantly undefined with the uPA-uPAR system mediating activity

25 proteolytic and cell viability.

Example 4. The genosuppression of uPA and uPAR with RNAi inhibits cell proliferation and induces apoptosis.

The effects of silencing of uPA and uPAR mediated by RNAi on cell proliferation and survival were examined with MTT analysis 72 h after transfection with specific shRNA for uPA and uPAR (FIG. 4A). The direction of the RNAi against uPA had no effect on the proliferative capacity of PC3 cells, while the RNAi specific for uPAR had a low inhibitory effect. On the contrary, a drastic reduction in the proliferation of PC3 cells with the RNAi was observed simultaneously to the direction of uPA and uPAR (FIG. 4A). The percentages of viable cells were reduced in the presence of uPAR siRNA and uPA-uPAR in approximately one

35 30% and 60% on average, respectively, compared to control cells. These results suggest that increasing levels of uPA and / or uPAR in tumor cells could give cells an increase in growth and survival capacity. As such, reducing levels of uPA and uPAR can induce apoptosis in cancer cells.

To examine this possibility, PC3 cells transformed with plasmid are expressed sh-uPA, sh-uPAR or shuPAuPAR. Molecular analysis of protein extracts from PC3 cells revealed sh-uPAuPAR transfection induced with proapoptotic genes, including Bax, Bcl-XS / L caspase 9 (FIG. 4B). In addition, staining with fluorescent dye of PC3 cells transfected with sh-uPAuPAR with FAM-VAD-FMK revealed an increase in caspase activity that was not detected in any of the simulated cells or cells transfected with

45 EV / SV (FIG. 4C and 4D). DNA fragmentation analysis provided additional evidence of apoptopic induction. As indicated in FIG. 4E, PC3 cells transfected with sh-uPAuPAR had DNA labeling, a common evidence of apoptosis, in agarose gel electrophoresis that was not detected in any of cells simulated or transfected with EV / SV. This DNA labeling was similar to that of apoptosis induced by treatment with actinomycin D (FIG. 4E), thus confirming that the observed cell death was a result of apoptosis. In addition, DNA labeling was not observed in cells transfected with either sh-uPA or sh-uPAR. These results are in accordance with the induction of caspase 9 and increased caspase activity in general as determined with FAM-VAD-FMK.

Example 5. The genosuppression of uPA and uPAR with RNAi inhibits its downstream signaling and tumorigenesis in nude mice.

The biological consequences due to the silencing of uPA and uPAR may be a result of changes in signaling mediated by uPA-uPAR and subsequent downstream functions. Since the increased expression of uPA and uPAR activates ERK1 / 2 signaling, the status of mitogen-activated protein kinases in uPA-uPAR genosuppression cells was examined. Immunoblot analysis shows that phosphorylation of ERK 1/2 was completely eliminated in cells transfected with sh-uPAuPAR, but not in control cells (FIG. 5A). ERK phosphorylation did not change in cells transfected with either shuPA or sh-uPAR. The total activity, including phosphorylation, of Stat 3 was basically suppressed in cells transfected with sh-uPAuPAR when compared to control cells (FIG. 5B). In addition, electrophoretic mobility shift assay (EMSA) with nuclear extracts from cells transfected with shuPAuPAR demonstrated that the genosuppression of the expression of uPA and uPAR inhibited the binding of extracts to the

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Stat 3 binding sites marked (FIG. 5C). Since the activation of Stat 3 contributes to the stimulation of the antiapoptotic pathway, the reduced level of phospho-Stat 3 as well as DNA binding activity may explain the increased susceptibility of PC3 cells transfected with sh-uPAuPAR to cell death. apoptotic Alternatively, constitutive ERK activation may contribute to cell survival.

5 It was investigated whether uPA-uPAR RNAi also suppressed the tumorigenicity of PC3 orthotopic tumors previously established in nude mice. PC3 cells were inoculated intraprostatically into the lateral lobe of the prostate. On days 7 and 14 after implantation, the tumors were injected with plasmid constructs expressing sh-uPA, sh-uPAR or sh-uPAuPAR. The mice were then sacrificed 14-15 days after the second dose of RNAi treatment, as needed with the morbidity resulting from the tumors that had formed in the control groups. The total morphology of primary tumors and metastatic sites was examined (FIG. 6A). No secondary tumors were reserved visually in mice treated with the sh-uPA, sh-uPAR and sh-uPAuPAR plasmids, while mice treated with EV, SV and simulated had secondary tumors in addition to the primary tumor within the prostate gland (FIG. 6A). Tumors are

15 dissected and weighed (FIG. 6B). An incidence of 90-100% of primary as well as secondary tumors was observed in groups treated with simulated, EV and SV. Although treatments with sh-uPA and sh-uPAR did not completely inhibit tumor growth, the weights of the tumor masses formed from these treatment groups were lower than the tumors of the control treatment groups (FIG. 6B) . A significant reduction in tumor weight shows mice treated with sh-uPAuPAR. Immunoblot analysis for protein levels in tumor samples confirmed that tumors treated with sh-uPAuPAR had significantly reduced levels of uPA and uPAR (FIG. 6C). An additional 150 µg of sh-uPAuPAR completely reversed the previously established prostate cancer.

Immunohistochemical analysis was performed on harvested tumor tissues embedded in paraffin to evaluate

25 the effects of sh-uPAuPAR on the behavior of PC3 cells in vivo. The expression of shuPAuPAR-directed RNAi did not change the general architecture of the prostate gland and staining with H and E showed a very normal histology, while staining revealed both tumor and host cells in the control groups. RNAi directed against either uPA or uPAR only slightly reduced tumor cells with respect to control groups. Supposedly, there was no in vivo rescue of uPA-uPAR RNAi-induced apoptosis. To test this, the paraffin embedded tumor sections of all treatment groups were stained for apoptopic markers using Klenow-FragEL DNA fragmentation analysis. This terminal marking of apoptotic cells demonstrates significant differences between treatment groups. The tumors of the groups treated with simulated, EV and SV showed low level staining, generalized for FragEL, therefore this indicates that the tumor cells were healthy. On the contrary, most areas

35 of PC3 prostate tumors treated with sh-uPAuPAR were positive for staining with Klenow.

Intratummoral co-injection of sh-uPA and sh-uPAR also resulted in an almost complete regression of previously established prostate tumor growth, while simulated control groups, EV and SV showed irreproducible and significant tumors (FIG. 7A and 7B). Immunoblot analysis demonstrated the selective genosuppression of the uPA and uPAR protein levels in tumors quoted with sh-uPA and sh-uPAR constructs (FIG. 7C). This co-treatment presented a very normal histology by staining with H and E while the tumors of the groups treated with simulated, EV and SV presented observable change in the general architecture of the prostate gland and staining with H and E showed both tumor cells and hosts (FIG. 7D). The genosuppression of uPA and uPAR by co-treatment also resulted in an induction

Significant death of apoptotic cells, as revealed by positive Klenow staining (FIG. 7E and 7F). When combined with the data presented in FIGS 6A-C and 7A-F, these results show that treatment with sh-uPAuPAR, however, has a potent RNAi effect when compared to co-treatment with sh-uPA and sh -uPAR, which causes a significant reduction in the size of the established tumor.

The results of in vitro DNA labeling analysis and an in vivo DNA fragment end labeling assay show that simultaneous genosuppression of uPA and uPAR with RNAi-based RNAi induces apoptosis. The downstream signaling mediated by uPA-uPAR is an excellent target for the treatment of hormone independent prostate cancer. UPA-uPAR-mediated downstream signaling is probably required for invasion, survival and cell proliferation in the PC3 cell line of prostate cancer.

55

Example 6. Functional signaling of uPA and uPAR and their role in tumorigenesis

The anomalous expression of uPA and uPAR was found to be one of the most frequent alterations in advanced stage prostate cancer. The fact that uPA and uPAR are overexpressed only in the advanced stage of prostate cancer suggests that uPA and uPAR influence the functional pathways that are relevant in determining the phenotypes of advanced-stage cancers, such as increased proliferation and invasion. Invasion through the extracellular matrix is a characteristic stage of tumor metastasis. The cancellation of the expression of either uPA or uPAR to suppress tumorigenesis has been achieved using several different approaches. The coupling of uPA with uPAR directs several different signaling molecules that form a unique network of 65 different types of biological responses, such as proliferation, migration, invasion, angiogenesis and metastasis. These biological responses to the uPA-uPAR binding appear to be highly specific for the

image12

cell type, the nature of the downstream signaling molecule and the level of its expression. The binding of uPA with uPAR activates ERK 1 and 2 and it is this induced ERK activity that is necessary for the migration of MCF-7 breast cancer cells induced by uPA. A signaling cascade that includes FAK, Src and She is responsible for the activation and cellular migration of ERK induced by uPA. On the contrary, the migration and proliferation of

5 vascular smooth muscle (VSMC) cells induced by uPA needed activation of the Stat pathway. In breast cancer cells, uPA-induced mitogenic activity requires activation of both Stat and ERK pathways. The antisense uPA inhibited PI3K / Akt signaling and sensitized the cells for apoptosis with staurosporin in the glioblastoma SNB 19 cell line. The union of uPA with uPAR probably activates signaling cascades to regulate cell migration, invasion, proliferation and survival.

The siRNA for uPA-uPAR in PC3 cells showed a remarkable suppression of invasion and proliferation as well as induction of apoptosis (FIGS. 3-4). Suppression of the uPA-uPAR system and downstream signaling molecules (ERK and Stat 3) was observed in cells transfected with sh-uPAuPAR PC3 but not in cells simulated or transfected with EV / SV (FIG. 5). This suggests that all the phenotypic changes observed in these cells are

15 mediated by the suppression of the interaction of uPA-uPAR and the phosphorylation state of its molecules downstream. UPA-uPAR signaling stimulates both Stat and ERK pathways and protects cancer cells from death. Several lines of evidence showed that both ERK and Stat 3 pathways are capable of protecting cells from apoptotic cell death. Transfection with either sh-uPA or shuPAR does not trigger apoptosis in PC3 cells (FIG. 4) and this may be because the blockage of either uPA or uPAR alone may not sufficiently influence ERK and Stat 3 molecules. downstream signaling.

Since uPA-uPAR signaling modulates ERK and Stat 3 expression, simultaneous inhibition of uPA and uPAR can alter these pathways, which leads to growth inhibition and induction of apoptosis. The uPA-uPAR system probably functions as a positive regulator of cell survival by facilitating

25 cell proliferation and survival, the two evidences of cancer. Therefore, when overexpressed in cancers, uPA and uPAR provide a cancer cell with increased proliferation and / or increased apoptosis resistance. In contrast, the genosuppression of the expression or function of uPA-uPAR should inhibit the growth of cancer cells and induce apoptosis. Stopping the signaling mediated by uPA-uPAR by means of genosuppression of uPA and uPAR simultaneously inhibited the growth of cancer cells and induced apoptosis. It should be noted, that the uPA-uPAR RNAi worked in the PC3 cell line of hormone-resistant prostate cancer. This suggests that the genosuppression of the expression of uPA-uPAR with RNAi is a strategy to inhibit the growth and survival of the hormone-resistant prostate tumor.

Orthotopic implantation of human cancer cells in athymic mice resembles

35 biological behaviors of these cells in humans, in particular with respect to the development of metastases. This has been shown to be true in particular for human prostate cancer cells, which form primary tumors and metastases with much less efficacy when implanted ectopically in athymic mice. An RNAi-based RNAi plasmid system represents a strategy that can effectively suppress uPA-uPAR expression in orthotopic prostate tumors as determined by immunoblot analysis (FIG. 6C). In addition, in vivo treatment of previously established orthotopic tumors with shi uPAuPAR-directed RNAi demonstrated an almost total inhibition of tumor growth, while only partial reduction with RNAi of either sh-uPA or sh-uPAR was observed (FIG. 6B). In addition, the co-treatment of orthotopic tumors previously established with sh-uPA and sh-uPAR also almost completely inhibited tumor growth (FIG. 7). No harmful effects were observed in animal groups

45 treated with RNAi compared to groups treated with simulated or with EV / SV. In addition, this approach can be directed to a wide variety of tumor types and inhibit uPA-uPAR-dependent malignant phenotypes in vitro as well as in vivo. Therefore, this RNAi system provides a new powerful therapeutic tool and also serves to analyze the downstream signaling fruits of uPA-uPAR as well as to offer treatment options for cancer intervention with clinical relevance. In addition, RNAi provides a convenient and selective way, new to interfere with the expression of uPA-uPAR and to study the biological significance of its signaling in cancer biology.

Despite advances in the understanding of the molecular mechanisms of human cancer, the development of therapeutic approaches for the clinical treatment of human malignancies remains a major challenge. The

55 genosuppression of the expression of uPA-uPAR significantly inhibited the growth of PC3 cells in vitro as well as in vivo and ultimately resulted in apoptopic cell death. Different target genes (ERK and Stat 3) were regulated downstream of the uPA-uPAR signal. PC3 cells had a low phosphorylation of Stat 3 and the phosphorylation of ERK was completely canceled when they were transfected with sh-uPAuPAR. RNAi induced uPA-uPAR cell death in PC3 cells in vitro as well as in vivo.

Example 7. Inverted 21 bp repeats driven by plasmid-based CMV promoter directed to uPAR and MMP-9 are processed to siRNA.

To determine whether the transcript driven by the CMV promoter (directed by uPAR and MMP-9) is processed

65 correctly to siRNA, SNB 19 cells will transfect a with control / EV, SV, puPAR, pMMP-9 and pUM. FIG. 8 illustrates a schematic representation of the construct. The cells were also transfected with an unrelated construct directed to GFP in cells other than GFP to determine the processing of the RNA to siRNA transcript, and confirm the fact that the results obtained are not simply degradation products of the target gene. Transfected SNB 19 cells other than GFP with pGFP resulted in the processing of RNA to siRNA transcript (FIG. 9A). Similarly, cells transfected with puPAR, pMMP-9 and pUM

image13

5 resulted in the processing of the RNA transcript to the appropriate siRNA. Cells transfected with EV will not produce any fragment similar to siRNA targeting uPAR or MMPs-9 indicating that the observed siRNA fragment is processed from the inverted repeat loops incorporated into the construct. Cells transfected with SV also produced no fragment similar to siRNA targeting uPAR or MMP-9; SV consisted of an imperfect inverted repeat sequence without homology with any known gene. When they were investigated with a sense oligo of 21 bp for SV, no DNA hybrid was observed: 21 bp RNA indicating that this builder did not profess fragments similar to siRNA.

SNB 19 cells transfected with pUM caused negative mRNA regulation of both uPAR and MMPs-9. To determine whether the plasmid construct containing sequences of 21 inverted base pairs 15 homologated to uPAR and MMP-9 would induce RNAi, SNF19 cells were transfected with control / EV, SV, puPAR, pump-9 and pUM. Total RNA was isolated from the transfected cells and the first strand of the cDNA was synthesized using a cDNA synthesis kit (Invitrogen). Next, the cDNA was subjected to PCR according to conventional protocols known to those skilled in the art. Using primers specific for uPAR, MMP-9, and GAPDH (see Table 1) in cells transfected with control / EV and SV, there was no reduction in the levels of uPAR or MMPs-9; while in cells transfected with puPAR and uPAR levels, mRNA was significantly reduced, and MMP-9 mRNA levels did not change. In cells transfected with pMMP-9, MMP-9 mRNA levels were reduced, while uPAR mRNA levels did not change indicating the specificity of vectors for target molecules. Cells transfected with pUM showed a decrease in mRNA levels of both uPAR and MMP-9. GAPDH levels did not change (FIG.

25 9B).

Example 8. Inhibition of MMP activity and uPAR protein levels with interfering RNA

SNB19 cells were transfected with EV / SV, puPAR, MMP-9 and pUM and then uPAR and MMP-9 levels in cell lysates were determined by Western blotting and gelatin zymography media respectively. SNB19 cells transfected with the pUM vector expressed decreased amounts of uPAR protein when compared to precursor cells and treated with EV / SV by Western blotting (FIG. 10A). To determine if this inhibitory effect was specific for uPAR, the levels of β-actin in the same transfer were evaluated. Β-actin levels were similar in all

35 streets confirming an equal load on all streets. Media conditioned from SNB19 cells infected with pUM significantly expressed low levels of MMP-9 activity compared to cells transfected with simulated and empty / encoded vector (FIG. 10B). MMP-2 levels did not change, indicating specific inhibition of the target protein. Quantitative analysis of the uPAR and MMP-9 bands by densitometry revealed a significant decrease in the uPAR protein (12 to 14 times) and the enzymatic activity of MMPs-9 (8 to 10 times) in cells transfected with pUM in comparison with precursor cells and transfected with EV / SV. Cells transfected with puPAR and pMMP-9 vectors inhibited the levels of uPAR and MMP-9 (FIG. 10A and 9B) in almost the same way as the bicistronic construct, but the negative regulation of the target molecules was more pronounced with the bicistronic construct compared to individual constructs.

45 Vector mediated expression of short hairpin RNA (shRNA) for uPAR and MUMP-9 can achieve efficient and stable genetic silencing in a glioma cell line in vitro and in vivo. RNAi-based genetic silencing can be adapted to direct overexpressed proteins in gliomas with significant therapeutic potential. Synthetic siRNA molecules also have the same suppression effect of endogenous MMP-9 uPAR levels.

Example 9. Inhibition of cell proliferation with siRNA for uPAR and MMP-9.

The MTT assay was used to assess the effect of siRNA vectors (EV / SV, puPAR, pMMP-9 and pUM) on the

55 proliferation of cells grown in microplates coated with vitronectin. After 3 days of infection, SNB19 cells infected with puPAR, puPAR, pMMP-9 and pUM vector showed a decrease in proliferation with respect to that of precursor SNB19 cells transfected with EV / SV (FIG. 11). The effect of the pUM vector was much higher in the proliferation of SNB19 compared to the individual siRNA constructs (puPAR and pMMP-9). There was no difference in proliferation between precursor SNB19 cells and transfected with EV / SV.

Example 10. The interference RNA inhibited the immunofluorescence of uPAR and MMP-9 and tumor-induced angiogenesis.

65 SNB 19 cells transfected with puPAR and pMMP-9 caused negative regulation of uPAR and MMP-9 protein levels as determined with immunocytochemistry. PUM transfected cells

image14

caused the negative regulation of both uPAR and MMP-9 protein levels as determined with immunocytochemistry (FIG. 12A). To determine the effect of the combined construct that expresses siRNA for both uPAR and MMP-9, SNB19 cells were transfected with puPAR, pMMP-9 and pUM; The cells were also transfected with EV and SV, which served as controls. From the results, it is clear that cells transfected with puPAR only had a negative regulation of uPAR protein levels. Cells transfected with pMMP-9 will have a negative regulation of MMP-9 alone, while cells transfected with pUM caused a negative regulation of uPAR and MMP-9 protein levels, indicating that the double construct was effective, if not more, to negatively regulate target protein levels. To test and the siRNA for uPAR and MMP-9 could also inhibit tumor-induced capillary formation, were SNB19 glioma cells transfected and not transfected with human endothelial cells. Immunohistochemical analysis was performed using factor VIII antigen to evaluate tumor-induced vessel formation in the in vitro coculture system and staining with H and E of these cocultures after transfection with EV / SV, puPAR or pMMP-9 and pUM. FIG. 12B shows that endothelial cells cultured with SNB19 cells formed networks similar to different capillaries in cultures transfected with simulated and empty vector in 24-48 h. Conversely,

15 SNB19 cells transfected with pUM did not induce the formation of capillary-like networks in endothelial cells. The quantification of the branching points and the number of branching was significantly reduced in cocultures transfected with pUM compared to the coculture transfected with precursor and empty / encoded vector (FIG. 12C). In addition, the effect was less than 50% in puPAR and pMMP-9 vector and was less than 50% in co-culture transfected with puPA and pMMP vector, when compared with the group treated with precursor EV / SV in relation to formation similar in structure to capillaries. Implantation of a chamber containing SNB 19 cells transfected with precursor EV resulted in the development of microvessels with fine, curved structures and many tiny bleeding points. on the contrary, implantation of SNB 19 cells transfected with the pUM vector did not result in the development of any additional microvessels (FIG. 12D).

Example 11. The siRNA for uPAR and MMP-9 inhibits the invasion of SNB19 cells.

Since the expression of siRNA inhibited uPAR and MMPs-9, its ability to inhibit cell invasion was evaluated. SNB19 cells transfected with EV / SV, puPAR, pMMP-9 and the PUM vector were allowed to invade through Matrigel coated filters. FIG. 13A illustrates that the staining of SNB 19 cells transfected with pUM was significantly lower than that of cells only thus transfected with EV / SV. Quantitative analysis of the cells showed that only 8% of the cells transfected with pUM invaded compared to the precursor cells and transfected with EV / SV (FIG. 13B). In addition, quantitative analysis of the invasion of SNB19 cells transfected with puPAR vector and pMMP-9 invaded 25% and 50% compared to precursor SNB19 cells transfected with EV / SV (FIG. 13A and 13B). RNAi also inhibited the invasion of SNB19 cells in a three-dimensional spheroid invasion model. FIG. 13C demonstrates that glioma spheroids transfected with simulated and empty / encoded vector bound to rat brain aggregates and progressively invaded the aggregates. However, co-cultures with glioma spheroids transfected with pUM failed to bind to rat brain aggregates and did not invade. Quantitative analysis indicated that only 2-4% of fetal rat brain aggregates remained in the precursor spheroid and transfected with EV / SV, while 90-95% of fetal rat brain aggregates remained in the spheroid. transfected with pUM (FIG. 13D). At 72 h, rat brain aggregates revealed approximately 25% and 45% invasion in cocultures transfected with puPAR and pMMP-9. Taken together, these findings provide strong evidence that silencing of uPAR and MMP-9 mediated with RNAi greatly inhibits the invasion of glioma cells in both in vitro models compared to the constructs of

45 individual siRNAs for uPAR and MMP-9. These results showed that the individual siRNA constructs for uPAR were more effective than the individual siRNA construct for MMP-9.

Example 12. Therapeutic effect of siRNA for uPAR and MMPs-9.

To assess the efficacy of the interference of the genetic expression of uPAR and MMP-9 mediated with RNAi in tumor evolution, the pUM vector was injected into tumor bearing mice using a stereotactic pump. To facilitate the detection of tumor and massive cells, human glioblastoma cells (SNB19) were evaluated with the cDNA for green fluorescent protein (SNB19-GFP). Microscopic examination of the brain sections revealed that control animals receiving PBS or empty vector (EV) only developed significant tumor growth after a 5-week follow-up period as visualized by GFP fluorescence and staining with H and E of similar sections. In contrast, tumor growth or fluorescence with GFP or staining with H and E was not detected in animals that received the pUM vector under the same conditions (FIG. 14A and 14B). The quantification of brain sections stained with hematoxylin and eosin or GFP sections with a neuropathologist who had been hidden from treatment did not reveal differences in tumor size between control groups and treated with empty vector; however, the total regression of the tumors was revealed in the group treated with pUM vector (FIG. 14C). In the case of an individual constructs treated with siRNA for uPAR and MMP-9, the previously established intracranial tumor growth was inhibited by 70% and 40%, respectively. Intraperitoneal injections of the pUM vector resulted in a complete regression of previously established intracranial tumor growth over a period of time.

65 longer than 6 months. These results demonstrated that the suppression of uPAR and MMPs-9 mediated with RNAi dramatically inhibited the growth of previously established intracranial tumor.

Inhibition of uPAR and MMP-9 mediated with RNAi can inhibit tumor growth in several independent ways. Apoptosis measured with DNA fragmentation was higher in the brains of animals injected with the stable clones of antisense uPAR compared to the precursor cell line. The antitumor effects observed in the intracranial tumor model could be due to the induction of cell death

5 tumor

Tumor cells depend on angiogenesis to survive and proliferate. Inhibition of uPAR and MMP-9 mediated with RNAi significantly inhibited tumor induction in an in vitro coculture system. The anti-angiogenic effects of the pUM vector suppressed the ability of tumor cells to recruit blood vessels necessary for survival and directed the anti-invasive effects on tumor cells themselves. The ability of uPAR siRNA to block tumor evolution could also include blocking the antiapoptotic and angiogenic effects of uPA. In general, no individual antiangiogenic agent (including angiostatin and endostatin) used as monotherapy in preclinical models is able to reduce the tumor burden after the tumors have reached 100 mm. It was reported that the absence of Plg, uPA, or tPA decreased from

The development of experimental choroidal neovascularization significantly compared to wild-type or uPAR-deficient mice. It was suggested that this effect was partially due to a modulation of the metalloproteinase activity of the matter. Although some studies have demonstrated the antiangiogenic effect of synthetic MMP inhibitors, virtually all of these inhibitors lack specificity for an individual MMP. For example, decreased vessel density and increased tumor cell apoptosis in primary tumors and metastases were observed in mice treated with KB-R7785, which inhibits MMP-1, -3 and -9. MMPIs have shown little clinical benefit when used as monotherapy in patients with advanced diseases. Therefore, the combined use of MMP inhibition with other modalities is also a strategy for cancer treatment.

Example 13. siRNA against uPAR and MMP-9 inhibits the level of phosphorylated ERK, MAPK and AKT.

The ERK, MAPK and AKT pathways play a major role in cell proliferation and survival. Western blotting was used to compare the levels of total and phosphorylated forms of ERK, MAPK and AKT using specific antibodies, specific for these molecules after transfection of SNB19 cells with EV / SV, puPAR, pMMP-9 and pUM. There were no significant differences in the amounts of total MAPK, ERK and AKT with constructs of EV / SV, puPAR, pMMP-9 and pUM (FIG. 15). However, the levels of the phosphorylated forms of MAPK, ERK and AKT decreased significantly with pUM compared to SNB19 cells transfected with EV / SV, puPAR and puPA (FIG. 15).

35 The binding of uPA has uPAR in MCF-7 cells activates ERK1 and ERK2, which are necessary in cell motility. In the prostate cancer cell line (PC3MLN4), hypoxia increased tumor cell invasion by positive regulation of uPAR expression, which could be mediated through the MAPK, ERK and p38 kinase signaling pathway. In addition, the positive regulation of the expression of uPAR with Bcl2 in hypoxia was mediated with the binding activity of SP1 DNA through the ERK signaling pathway. In the absence of EGFR, an alternative route links uPAR to ERK. However, this route is silenced with the expression of EGFR, which therefore indicates the involvement of uPAR in cell motility. Stable transfection of PTEN (phosphatase and tensin homolog) reduced MMP-9 secretion caused by focal adhesion kinase phosphorylation and ERK1 / ERK2 signaling induced by hyaluronic acid. Glioblastomas with EGFR VIII amplification demonstrated the highest levels of MMP-9. Transient transfection of SNB 19 cells with mt ERK or mt JNK repressed the

45 MMPs-9 promoter suggesting that interference with any route could result in the inhibition of MMP-9 expression. The regulation of MMP-9 activation with various stimuli or in different cellular scenarios may involve different signal transduction pathways. ERK inhibition with specific MEK inhibitors blocked the expression of MMP-9 in breast cancer cells and decreased the production of MMP-9 and attenuated the invasion capacity in vivo in squamous cell carcinoma cells of the head and neck. The stable transfected cells, mt-ERK, were less invasive and significantly reduced MMP-9 levels. It has been reported that these two signaling pathways (MAPK and ERK1 / 2) are activated when uPA joins uPAR. The pUM construct inhibits the phosphorylated forms of these signaling pathway molecules (FIGS. 1516).

55 Example 14. Therapies for medium-sized cancer with RNAi and administration of siRNAs

Inhibition of the siRNA of genes such as, for example, overexpression of uPAR and MMP-9 extends the list of therapeutic modalities available for the treatment of human cancer. Although some antisense approaches are available, including oligonucleotide and ribose antisense technologies, their efficacy is not satisfactory. Inhibition of uPAR and MMP-9 mediated by RNAi completely suppressed the tumor growth of glioma previously established in athymic mice. Therefore, RNAi is a more powerful alternative with respect to other genetic tools such as oligonucleotide and ribose antisense technologies in reducing the expression of target genes. The effects of RNAi or similar to those of RNAi were more potent than antisense effects in reducing the expression of target genes, which also suggest the potential application capacity of RNAi. A peptide vector was used that included arginine-glycine-aspartic acid tumor-seeking motif in a cyclic conformation, a DNA binding oligo lysine and histidyl moieties to facilitate

image15

administration in the cytosol. The peptide vector can function as an siRNA vehicle. RNAi-based gene therapy is a new approach to the treatment of gliomas and other metastatic tumors, which include prostate cancer, breast cancer, and melanoma.

5 Example 15. Effect of the pCU vector on cathepsin B and uPAR protein levels in total cell extracts.

RNAi directed against proteolytic degradation is an intervention to prevent the invasion of cancer cells (FIG. 17). Cathepsin B and uPAR have been shown to play significant roles in ECM degradation. Transfection of SNB19 cells with the siRNA expressing vector for cathepsin B and uPAR (pCU) greatly inhibited the expression of both proteins compared to simulated and empty vector (EV) controls (FIG. 18 A and C). The levels of β-actin determined that equal amounts of protein were loaded into the gel (FIG. 18). Quantitative analysis of cathepsin B and uPAR bands by densitometry revealed a significant decrease (P <0.001) of cathepsin B protein (14 to 16 times) and uPAR (10 to 12 times) and in

15 cells transfected with pCU compared to cells transfected with simulated and empty vector (FIG. 18B and D). Cells transfected with pU and pC vectors inhibited the levels of uPAR and cathepsin B, respectively (FIG. 18 A and C).

Example 16. Inhibition of capillary network formation induced by tumor cells with pCU vector.

Emerging tumors depend on the formation of new blood vessels that provide fuel for tumor growth. Since cathepsin B and uPAR have been reported to regulate angiogenesis, the effect of pCU on tumor-induced angiogenesis was evaluated. Immunohistochemical analysis was performed using factor VIII antigen to evaluate tumor-induced vessel formation in an in vitro coculture system and staining with H and E 25 for cultured endothelial cells in the presence of conditioned media from SNB 19 cells after transfection with simulated, empty vector, pC, pU or pCU. The results demonstrate that endothelial cells form capillary-like structures in the presence of cells transfected with simulated and empty vector within 48 h; while the pCU vector significantly inhibited the formation of tumor-like networks induced by tumor cells (FIG. 19A). The quantification of the branching points and the number of branches was undetectable in cocultures transfected with pCU compared to simulator and empty vector (FIG. 19B). In addition, the effect was less than 50% in cocultures treated with pC or pU when compared to pCU vector in relation to capillary-like structures. To confirm in vitro coculture experiments, it was examined whether the pCU vector can inhibit tumor angiogenesis in vivo as evaluated with the dorsal chamber model. The implanted chambers containing SNB19 cells transfected with simulated and empty vector (EV resulted in

35 development of microvessels (as indicated by arrows) with fine curved structures and many bleeding points and minutes. In contrast, the implanted cameras of SNB19 cells transfected with the pCU vector did not result in the development of additional microvessels (FIG. 19C).

Example 17. Inhibition of the migration of spheroids of SNB19 with siRNA.

To determine whether the expression of cathepsin B siRNA and uPAR is capable of influencing the migration and proliferation of tumor cells, SNB 19 spheroids were transfected with the pCU vector. As shown in FIG. 20A, much higher cell migration occurred from the spheroid transfected with simulated and empty vector (EV) and up to 50% inhibition of admiration was observed with spheroid transfected with

45 individual construct. However, cell migration of tumor spheroids was completely inhibited in spheroids transfected with the pCU vector. Migration of spheroid transfected with simulated and empty vector was significantly higher (P <0.001) compared to spheroids transfected with pC, pU and pCU as quantified with the number of cells that migrate out of the spheroid (FIG. 20B). The migration of cells from the spheroids was inhibited with bicistronic construct compared to individual RNAi constructs for these molecules. A few cells migrated from the SNB19 spheroids transfected with pCU compared to those in the simulated and empty vector controls, thus indicating the role of cathepsin B and uPAR in cell migration.

Example 18. The siRNA against cathepsin B and uPAR inhibits the invasion of tumor cells.

55 To assess the impact of cathepsin B inhibition and uPAR mediated with siRNA on glioma invasion capacity, a model system was used. SNB19 cells transfected with simulated and empty vector extensively invaded the Transwell inserts coated with Matrigel as seen with intense staining of the cells. In contrast, cultures transfected with pC, pU and pCU had less invasion capacity through the reconstituted basement membrane, compared to cells transfected with simulated and empty vector (FIG. 20C). The quantitative invasion determination confirmed that SNB19 cells transfected with the vector pC, pU and pCU invaded only 55%, 40% and 6% respectively compared to controls transfected with simulated and empty vector (FIG. 20D). Inhibition of the invasive behavior of these cells as determined by the Matrigel invasion assay was much higher in those in the cells.

65 transfected with bicistronic construct when compared to the individual construct.

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The extent of the effect of pCU in spheroid invasion assay was tested. In the spheroid coculture assay, control glioma spheroids and spheroids transfected with the empty vector progressively invaded the brain aggregates of fetal rats and resulted in a partial to almost complete inhibition of the invasion of spheroid transfected with pCU (FIG. 20E). The quantification of aggregates of

5 fetal rat brain revealed that glioma spheroids invaded fetal rat brain aggregates by 25% in 24 h,> 70% in 48 h and> 90% at 72 h. In contrast, tumor spheroids transfected with the pCU vector did not invade fetal rat brain aggregates. At 72 h, rat brain aggregates revealed an invasion of approximately 90%, 85%, 55% and 35% in co-cultures transfected with simulated, empty vector, pC, and pU, but only an invasion of one 2% to 3% in cocultures transfected with pCU (FIG. 20F). Taken together, these findings provide great evidence that the silencing of cathepsin B and uPAR mediated with RNAi greatly inhibits the invasion of glioma cells in both in vitro models, and that the effect was much greater with bicistronic construct compared with individual constructs.

The acquisition of the invasion capacity of tumor cells is one of the aspects of the evolution of the tumor.

15 There are several reports that indicate the expression of cathepsin B and uPAR are essential components of the invasion process. Transfection with the pCU vector inhibited the invasion capacity of SNB 19 and steroid cells in the co-culture of invasion assays in Matrigel and spheroids. The requirement for cathepsin B invasion into Matrigel could be due to its interaction with a protease network. Cathepsin B was shown to activate serine proteinase precursors to their active forms, such as pro-uPA and metalloproteinases, such as prostromelysin. The ability to invade through Matrigel of transformed human breast epithelial cell lines was related to the expression of cathepsin B and was inhibited with cysteine proteinase inhibitors. In ovarian cancer cells, the inhibition of cell surface cathepsin B prevents the activation of pro-uPA, and subsequently, the invasion of carcinoma cells through Matrigel. The activity of cathepsin B in human colon cancer is associated with the invasion capacity of cancer cells, cells

25 endothelial and inflammatory cells as well as apoptotic and necrotic cell death. It is known that uPA and uPAR are overexpressed in various neoplasms that include breast, ovarian and gastric cancers, and have been shown to be essential in maintaining invasive and metastatic phenotype.

Example 19. Negative regulation mediated with siRNA of Cathepsin B and uPAR reduces proliferation of SNB cells 19.

A conventional MTT assay was used to evaluate the effect of siRNA vectors (Control, EV, SV, pC, pU and pCU) on the proliferation of cells grown in vitronectin-coated microplates (FIG. 21). 72 h after infection, SNB 19 cells infected with pC, pU and pCU vector showed a decrease in proliferation

35 with respect to that of SNB19 and vector controls. Cells transfected with PCU did not show any appreciable growth even after 7 days of transfection. No floating cells or cell debris were observed in any of the transfected cells even after 7 days of testing indicating the absence of apoptosis.

Example 20. The negative regulation mediated with siRNA of uPAR and cathepsin B inhibits the phosphorylation of ERK1 / 2 and FAK.

To determine the effect of the negative regulation of uPAR and cathepsin B on signaling pathway molecules, the phosphorylation of ERK and FAK was tested with Western blotting both of which are

45 directly involved in the survival, migration and proliferation of tumor cells. FIG. 22 show that the simultaneous negative regulation of uPAR and cathepsin B mediated by RNAi delays the phosphorylation of ERK1 / 2 and FAK and the effect was much less with individual constructs.

The results demonstrate that the negative regulation of uPAR and cathepsin B induces the negative regulation of the phosphorylation of ERK1 / 2 and FAK that are directly responsible for cell survival and proliferation. The involvement of uPAR in the ERK-FAK cascade has been previously reported in human carcinoma HEp3 cells, the role of cathepsin B is still unclear. A negative regulation of combination of uPAR and cathepsin B is more effective in inhibiting the phosphorylation of ERK1 / 2 and FAK.

Example 21. The siRNA of cathepsin B and uPAR suppresses the growth of intracranial tumor.

An intracranial tumor model was used to assess the potential effects of RNAi-mediated inhibition on tumor growth previously established in vivo. The brain sections of the untreated (simulated) and EV treated control groups were characterized by a large dispersion of tumor growth by staining with H and E and high fluorescence with GFP after a 5-week follow-up period (FIG. 23A and B). However, GFP fluorescence was not detected in the brain sections of mice treated with the pCU vector (FIG. 23A and B). The additional quantification of the brain sections stained with H and E punctuated by a neuropathologist whose treatment was hidden did not reveal a difference in tumor size between the simulated and empty vector treatment groups and a significant growth regression of the tumor in 55% and 65% in 65 in the groups treated with pC and pU compared to controls. However, the total regression of previously established tumors was revealed in the group treated with pCU (FIG. 23). These results demonstrate

image17

that suppression of cathepsin B and uPAR mediated with RNAi significantly inhibited the growth of intracranial tumor.

Local intracranial administration of pCU using mini osmotic pumps effectively inhibited growth

5 of the human malignant glioma. Mini osmotic pumps maintain a well defined and consistent drug exposure pattern for a significant period of time and can be used successfully to deliver agents to the brain. Negative regulation of cathepsin B and uPAR results in the inhibition of tumor-induced angiogenesis. An in vitro coculture assay was used to test the effect of pCU on angiogenesis. The results demonstrate that cathepsin B and uPAR play relevant roles in stimulating angiogenesis, suggesting a possible mechanism of action for the antitumor activity of pCU in vivo in the intracranial tumor model. An intense staining for cathepsin B is present in the endothelial cells of the neo-vessels but not in the microvasculature previously existing in the prostate. Similarly, strong immunostaining of cathepsin B was observed in rat brain microvascular endothelial cells as capillary tubes formed in vitro. Since cathepsin B is shown to be a

15 TIMP and TIMP inhibitors are inhibitors of angiogenesis, cathepsin B may also stimulate angiogenesis, which has a relevant role in the spread of the tumor. The direction of cathepsin B and uPAR mediated by RNAi suppressed the growth of the previously established intracranial tumor, possibly by inhibiting angiogenesis and invasion capacity. These results also support that the negative regulation of the expression of siRNA-mediated target genes is stable enough within the brain microenvironment.

Example 22. Effect of siRNA constructs on uPAR protein, and enzymatic activity of uPA and MMP-9 in glioblastoma SNB19 cells.

25 To simultaneously inhibit three endogenous genes with hairpin siRNAs, a vector (pU2M) was constructed that expressed siRNA for uPAR (77-98 bases of human uPAR on RNA), uPA (346-367 bases of human uPA on RNA) and MMP-9 (360-381 bases of human MMP-9 in RNA) under the control of the CMV promoter (FIG. 24). Bases indicate positions in a full length coding sequence. Western blot analysis was performed to examine the effect of transfection of empty vector / encoded vector (EV / SV), puPAR, puPA, MMP-9 and pU2M at concentrations of uPAR protein in SNB 19 cells. The band of the uPAR protein was present in SNB19 cells transfected with EV / SV, puPA and pMMP-9, while significantly reduced in cells treated with puPAR and pU2M (FIG. 25A). The effect of the tricistronic construct (pU2M) was greater than that of puPAR (FIG. 25A). GAPDH levels determined that equal amounts of protein were loaded into the gel (FIG. 25A). Fibrin zymography was performed to examine the effect of treated SNB 19 cells

35 with EV / SV, puPAR, puPA, pMMP-9 and pU2M in the enzymatic activity of uPA. Gelatin zymography was performed to determine the effect of these constructs on MMP-9 levels in SNB19 cells. MMP-9 levels were significantly reduced in SNB19 cells treated with puPAR, pMMP-9 and pU2M compared to precursor cells, treated with EV / SV and puPA (FIG. 25B). Interestingly, MMP-2 levels were also regulated negatively in cells treated with pU2M. Care was taken to load equal amounts of protein. (FIG. 25B). The enzymatic activity of uPA (MR 55 000) was significantly reduced in cells treated with puPA and pU2M compared to the precursor groups, treated with EV / SV, puPAR and pMMP-9 (FIG. 25C).

The effect of the tricistronic construct was more pronounced than the individual siRNA constructs for these

45 molecules Determined with immunohistochemical analysis, transfection of puPAR, puPA, pMMP-9 and pU2M decreased the concentrations of uPAR, uPA and MMP-9 in SNB 19 cells. FIG. 25D shows the protein levels of uPAR, uPA and MMP-9 in precursor cells, cells transfected with EV / SV, puPAR, puPA, pMMP-9 and pU2M using antibodies specific for uPAR, uPA and MMP-9. The respective intensities of uPAR, uPA and MMP-9 were high in precursor cells and in cells transfected with EV / SV. In contrast, the intensity of uPAR decreased in SNB19 cells transfected with puPAR and pU2M. Transfection of puPA and pU2M significantly decreased the intensity of the uPA protein compared to precursor cells, cells transfected with EV / SV, puPAR and pMMP-9. In addition, the concentration of MMPs-9 protein decreased significantly in cells transfected with pMMP-9 and pU2M compared to precursor cells, cells transfected with EV / SV, puPA and puPAR. These results demonstrate that the effect of individual constructs

55 is molecule specific and that the effect of the tricistronic construct is much more pronounced than that of the individual constructs alone.

Example 23. puPAR, puPA, pMMP-9 and pU2M inhibit the formation of tumor-induced capillary networks.

The growth of a glial tumor depends on the induction of new capillary blood vessels that are necessary to support the development of tumor mass. A coculture system was used in which microvascular endothelial cells were induced with conditioned media from glial cells to form capillary-like structures to examine the effect of suppression of uPAR, uPA and MMP-9 mediated by RNAi. Immunohistochemical analysis using factor VIII antigen to evaluate tumor-induced vessel formation 65 co-culture system in vitro and staining with H and E. Endothelial cells form capillary-like structures in the presence of precursor SNB19 cell conditioned media and transfected with EV / SV (FIG.

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26A). In contrast, transfection of SNB 19 cells with vectors that express siRNA for uPA, uPAR and MMP9 either individually or in combination partially or totally inhibited tumor-induced microvessage formation (FIG. 26A). No new branching points or / or an increase in the number of branches were detected in cells transfected with pU2M compared to cells treated with EV / SV (FIG. 26A). In addition, compared

5 with cells treated with EV / SV, the formation of capillary-like structures was inhibited by ~ 5 5% in cultures treated with puPAR, ~ 36% in cultures treated with puPA and ~ 60% in cultures treated with pMMP-9 (FIG .26B).

The conditioned medium from a line of glioblastoma cells transfected with pU2M inhibited the simulated structures or empty vector (FIG. 26A-B). This indicates that the angiogenic signal necessary for the induction of angiogenesis was not present in cells transfected with pU2M. As shown by the absence of angiogenic induction in glioma SNB19 cells transfected with pU2M, the negative regulation of uPA, uPAR and MMP-9 by hRNA caused the negative regulation of angiogenic factors. The absence of uPA or tissue-type plasminogen activator (tPA) significantly decreased the development of experimental choroidal neovascularization compared to wild-type (WT) or uPAR-deficient mice (uPA - / -). Be

15 has reported that there was a significant decrease in the growth of the primary tumor in uPA - / - and in mice deficient in plasminogen activating inhibitor 1 (PAI-1 - / -), with respect to WT mice and tumors in uPA - / - and PAI - / - mice they had lower proliferation rates and higher apoptotic and also had a different neovascular morphology, compared to WT mice. Several peptides that have been shown to inhibit uPA binding with bacteriophage presentation inhibit angiogenesis and primary tumor growth in syngeneic mice.

Example 24. puPAR, puPA, pMMP-9 and pU2M inhibit invasion in SNB19 cells.

Proteolytic degradation of ECM components is relevant for tumor cell invasion. For

To assess the impact of inhibition of uPAR, uPA and MMP-9 mediated by siRNA on glioma invasion capacity, two models were used. In the first model, the invasion capacity of SNB19 cells transfected with puPAR, puPA, pMMP-9 and pU2M was compared with those infected with the EV / SV vector. SNB19 cells transfected with EV / SV and precursor cells demonstrated extensive invasion through Transwell inserts coated with Matrigel, as indicated by intense cell staining. In contrast, cultures transfected with puPAR, puPA, pMMP-9 and pU2M were less invasive through the reconstituted basement membrane, as indicated by staining intensity compared to controls (FIG. 26C). quantification confirmed that transfection with puPAR, puPA, pMMP-9 and pU2M vectors reduced invasion with SNB 19 cells by 9%, 40%, 15% and 2%, respectively, compared to precursor controls and transfected with EV / SV (FIG. 26D). Inhibition of invasion was higher in cells transfected with the

35 tricistronic constructor when compared to individual constructs alone.

The effect of the puPAR, puPA, pMMP-9 and pU2M vectors was examined using a spheroid invasion assay. A potential, significant advantage of the use of glioma spheroids is that tumor cells that grow in three-dimensional cultures have properties that are more similar to those of tumors in vivo. In the spheroid co-culture system, control spheroid and spheroid transfected with the EV / SV vector progressively invaded fetal rat brain aggregates while spheroids transfected with puPAR, puPA, pMMP-9 and pU2M demonstrated inhibition of invasion from partial to almost complete (FIG. 27A). The quantification revealed that glioma spheroids invaded fetal rat brain aggregates by 30% in 1 day, 55% in 2 days and 95% in 3 days, at which time the tumor spheroid and rat aggregates be

45 had combined into a single entity (FIG. 27B). A similar trend was observed with glioma spheroids transfected with the EV / SV vector. In contrast, tumor spheroids transfected with the pU2M vector did not invade fetal rat brain aggregates. At 3 days, rat brain aggregates were invaded by approximately 96%, 95%, 45%, 25% and 15% in the precursor cocultures, transfected with EV / SV, puPA, pMMP-9 and puPAR, and 1% in cocultures transfected with pU2M (FIG. 27B). These results provide strong evidence that pU2M inhibits glioma invasion in both in vitro models.

Example 25. The pU2M completely reverses the growth of the intracranial tumor.

The negative regulation of the levels of uPAR, uPA and MMP-9 was examined using any of constructs

55 individual or tricistronic causes the regression of intracranial tumor growth established previously in athymic mice. All animals in the control groups and treated with EV / SV had intact brain tumors that were characterized by strong fluorescence with GFP (FIG. 27C) while the brain sections of mice treated with puPAR, puPA and pMMP-9 they had small tumors, illustrated with a fluorescence with minimal GFP. In particular, GFP fluorescence was not detected in brain sections of mice treated with pU2M (FIG. 27C). The additional quantification of these sections (scored by a neuropathologist whose treatment conditions are hidden) revealed no difference in tumor size between the groups treated with precursor and with EV / SV and a significant regression of established intracranial tumor growth. previously in two groups treated with puPAR, puPA and pMMP-9 (80%, 55%, and 68% respectively) compared to the control groups (FIG. 27D). However, the complete regression

65 of the previously established intracranial tumor growth was revealed in the group treated with pU2M. These results demonstrate that the suppression of uPAR, uPA and MMP-9 mediated with RNAi using a construct

Tricistronic will completely eradicate the growth of malignant glioma in nude mice.

Example 26. Inhibition of ERK1 / 2 phosphorylation.

5 To better understand the effect of the negative regulation of uPAR, uPA and MMP-9 mediated with siRNA in signaling pathways, the total and phosphorylated levels of ERK1 / 2, which are directly involved in survival, were tested, Migration and proliferation of tumor cells. Western blots showed that there was no significant difference in the total concentrations of ERK1 / 2 in control cells and transfected with EV / SV compared to cells transfected with puPAR, puPA, pMMP-9 and pU2M (FIG. 28). However, the concentration of phospho-ERK1 / 2 was significantly reduced in SNB19 cells transfected with the pU2M vector compared to control SNB19 cells, transfected with EV / SV, puPAR, puPA and pMMP

9. In particular, there was no effect on phospho-ERK levels in SNB19 cells transfected with any of the individual constructs. GAPDH levels indicated that equal amounts of protein were loaded into the gel (FIG. 28).

Example 27. Expression of MMP-9 protein and cathepsin B minor of gene specific siRNAs in a glioma cell line.

To test the efficacy of the simultaneous inhibition of two endogenous genes with a hairpin siRNA expression vector, a vector that expresses siRNA for cathepsin B (732 to 753 bases of human cathepsin B mRNA) and MMP was constructed. -9 (360 to 381 bases of human MMP-9 mRNA) under the control of the human cytomegalovirus (CMV) promoter (pCM) (FIG. 29). Bases indicate positions in a full length coding sequence. FIG. 30A demonstrates that transfection with pC and pCM vector specifically inhibited cathepsin B levels compared to simulated controls, vacuum, and pM vector. The levels of 25 β-actin evaluated in the same transfer indicated that the inhibition of cathepsin B was specific and confirmed the same sample load. MMP-2 and MMP-9 levels were determined in the conditioned medium in the transfected cells. The amount of MMP-9 released from cells transfected with simulated and empty vector (EV) were the same. Cells transfected with pM and pCM vector expressed low levels of MMP-9 compared to simulated controls, EV, and pC. There was no change in MMP-2 expression demonstrating the specific inhibition of the pM and pCM vector sequence (FIG. 30B). To confirm that the decrease in MMP-9 activity was due to a decrease in protein expression, the conditioned medium was analyzed using immunoblotting with an antibody specific for MMP-9. The MMP-9 protein band decreased dramatically with immunoblotting of the conditioned medium of cells transfected with pM and pCM vector, but the bands were significantly much higher in the medium.

Conditioning of infected cells with the empty vector or with the pC vector (FIG. 30C).

Example 28. Inhibition of capillary network formation induced by tumor cells with PCM vector.

The growth of a glial tumor depends on the induction of new capillary blood vessels as they are necessary to support the development of tumor mass. A coculture system was used in which microvascular endothelial cells were induced with glial cells to form capillary-like structures to examine the suppression of cathepsin B and MMP-9 mediated by RNAi. SNB19 induced endothelial cells for differentiation in capillary-like structures in 72 h. In contrast, transfection of SNB 19 cells with the siRNA expressing vector for cathepsin B and MMP-9 completely inhibited the morphogenesis of microvessels

45 induced by tumor cells (FIG. 31A). The additional quantification of the branching points and the number of branches was undetectable in cocultures transfected with pCM compared to simulator and empty vector (FIG. 31B). In addition, the effect was only 50% in cocultures treated with pC or pM when compared with pCM vector in relation to capillary-like structures. To confirm the in vitro co-culture experiments, it was examined whether the pCM vector could induce tumor angiogenesis in vivo as evaluated with the dorsal window model. Implantation of the chamber containing SNB19 cells transfected with simulated and empty vector (EV) resulted in the development of microvessels (as indicated by arrows) with curved fine structures and many tiny bleeding points. In contrast, implantation of SNB 19 cells transfected with the pCM vector did not result in the development of additional microvessels (FIG. 31C).

55 Maintaining the growth of malignant tumors is closely related to the development of the vascular network that provides nutrients to the tumor. The formation of a vascular network characterized by closed polygons and structures similar to complex meshes in cells treated with the pCM vector was not observed. Usually, this network is observed when glioma cells are cocultured with endothelial cells. The proteolysis of extracellular matrix components allows endothelial cells to migrate and releases angiogenic signaling molecules stored from the extracellular matrix. Immunohistochemical analysis showed that cathepsin B was highly expressed in astrocytomas and malignant anaplastic glioblastomas compared to normal brain tissue.

These results show that MMPs can stimulate angiogenesis and the absolute lack of MMP activity can prevent the formation of new blood vessels. The tumor regression achieved with treatment 65 combined in the present disclosure is due to the complementary actions of cathepsin B and MMP-9. The direction of the expression of cathepsin B and MMP-9 in tumor cells is an effective approach to control

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angiogenesis and tumor growth.

Example 29. Suppression effects of the pCM vector on migration and invasion of glioma

5 Cell migration requires coordinated regulation of cell-cell junctions, cell-matrix junction and matrix remodeling. The influence of the suppression of cathepsin B and MMP-9 on the ability of cells to migrate in vitronectin in a spheroid migration assay was studied. Multicellular glioma spheroids were cultured from SNB19-GFP cells in 6-well plates coated with agarose. After checking the viability using morphology and trypan blue exclusion, spheroids of similar diameter (100200 µm) were transfected with simulated, empty vector (EV) or pCM vector expressing siRNA for cathepsin B and MMP-9. Three days later, the individual spheroids were placed on vitronectin-coated plates and allowed to migrate. FIG. 32A indicates that the cells of the control spheroids and the spheroids infected with the empty vector showed a significantly higher ability of the cells to migrate compared to the cells infected with the pCM vector. Proteolytic degradation of the components of the

Extracellular matrix is relevant for tumor cell invasion. To investigate whether siRNA expression for cathepsin B and MMP-9 plays a role in glioma invasion capacity, the invasion capacity of transfected SNB19 cells with the pCM vector was compared with respect to the infected cells with simulated and vector empty. SNB19 cells transfected with simulated and empty vector (EV) invaded through Matrigel more extensively compared to cells transfected with the vector pCM vector that penetrated through the matrigel (FIG. 32B).

The extent of suppressive effects of siRNA in a spheroid invasion model was examined. Happy country in a potential advantage of the use of glioma spheroids is that it has been shown that tumor cells that grow in three-dimensional cultures inhibit properties that closely resemble those of tumors in vivo. The

25 spheroid transfected with simulated and empty vector invaded 25% of normal brain aggregates in one day, 50% in every day and in three days, 95% of tumor spheroid and brain aggregate had combined in one single entity (FIG. 32C). In contrast, glioma spheroids transfected with the pCM vector that expresses siRNA for cathepsin B and MMP-9 remained separate from normal brain aggregates.

The present disclosure shows that the expression of siRNA directed by the CMV promoter against cathepsin B and MMP-9 (pCM) can satisfactorily silence the expression of cathepsin B and MMP-9 in the glioblastoma SNB19 cell line, such as analyzed by Western blotting and gelatin zymography. The results also demonstrate that the invasive potential of glioma cells treated with the pCM vector was significantly inhibited. Cancer cells must be separated from neighboring cells and from

35 components of the extracellular matrix to migrate and invade. Proteolysis can directly modulate adhesion to the cell matrix either by removing the adhesion sites or by exposing a binding site, which in turn can influence cell migration. Inhibition of cathepsin B and MMP-9 mediated with RNAi significantly blocked the migration of glioma SNB19 cells as shown in a spheroid migration assay.

Example 30. RNAi induces complete regression of glioblastoma tumors in nude mice.

The ability of siRNA for MMP-9 and cathepsin B to inhibit the regression of intracranial SNB19 tumors was tested in athymic mice. Mice with previously established glioma growth were injected stereotacticly with PBS (simulated), empty vector (EV), pC, pM and pCM vector. The brain sections of simulated and EV treated mice exhibited rapid tumor growth while mice injected with the pCM vector using mini osmotic pumps in a previously established tumor resulted in a complete inhibition of tumor growth over a period of time. 5 weeks (FIGS. 32A and 32B). The quantification of the tumor size showed a total reversion of the tumor in the group treated with pCM vector compared to the simulated or empty vector (FIG. 33C). The brain sections of mice treated with the group treated with pC or pM vector, resulted in a tumor regression of approximately 50% compared to the control groups. Intraperitoneal injections of the vector also resulted in a complete regression of previously established intracranial tumor growth without indication of tumor cells over a long period of several months (FIG. 33D). Therefore, the RNAi was able to

55 Completely eradicate the growth of the malignant glioma tumor in this athymic mouse model. Sustained suppression of glioma growth could be due to amplification of siRNA. SiRNA against cathepsin B and MMP-9 eliminates glioma growth more effectively than antisense oligodeoxynucleotide for MMP-9 and cathepsin B. Therefore, the control of both cathepsin B and MMP-9 expression has considerable significance for the regulation of tumor progression.

The anti-cancer efficacy of the RNAi-mediated inhibition of cathepsin B and MMP-9 was demonstrated. Comparison of antisense oligonucleotide suppressive effects and siRNAs directed against the same targets in mammalian cells revealed that the IC50 value for siRNA was approximately 100 times less than that of antisense oligonucleotides. The ability of siRNA to silence sequence specific target genes and

65 lower concentrations necessary to inhibit genetic expression make RNAi a powerful tool for genetic therapy.

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To develop a vector capable of producing hairpin siRNA molecules for uPA and uPAR, the mammalian expression plasmid pCDNA 3 vector was used. FIGS. 34-35 illustrate schematic representations of the 5 different forms of RNAi constructs directed by U6 and CMV. Autocomplementary inverted repeat sequences spaced with a 9 base G and C deficient region directed to uPA (346 to 367) and uPAR (77 to 89) were synthesized. The oligos for uPA were terminated with HindIII sites and the oligos for uPAR were terminated with BamHI and self-hybridized by heating at 100 ° C for 5 min and cooled to room temperature in 6x SSC which resulted in the formation of double-stranded DNA molecules with the respective ends of adhesive restriction sites. These dsDNA molecules were ligated to the BamHI and HindIII sites of plasmid vector pCDNA3, resulting in the formation of a plasmid containing inverted repeats for uPA and uPAR downstream of the CMV promoter and terminated with a BGH terminator. The resulting plasmid, called pU2, when transfected into mammalian cells resulted in the production of a double hairpin siRNA molecule that targeted both uPA and uPAR that were further processed.

15 with an RNAds that recognized enzymes (DICER) to produce individual siRNA molecules to induce RNAi. A sequence homologous to GFP was used in the construction of an encoded vector. Imperfect sequences were used, which do not form a perfect hairpin structure to develop the encoded vector. Two self-complementary oligos were synthesized and hybridized to generate a molecule of ADNds with HindIII sites. This dsDNA molecule was ligated into the HindIII site of the plasmid of pCDNA3. The resulting plasmid was called pSV. The resulting CMV-directed transcript had no hairpin structure and was not homologous with any native gene.

An expression cassette expressing siRNA for uPA and uPAR was subcloned into the Ad5 shuttle vector in the ∆E1 region directed with any of a pol II RNA or pol III RNA promoter. The resulting plasmid was co-transfected.

25 with Ad 5 genomic plasmid (such as PJM17) in 293 permissive replication cells to generate Ad 5 virus particles with recombinant replication deficit containing siRNA expression cassette for uPA and uPAR (FIG. 36). A GFP RNAi vector was constructed to determine the specificity of the address using RNAi. Stable SNB19 cells expressing GFP were used as controls and transfected with RNAi directed against GFP. Cells transfected with GFP RNAi lost GFP expression (FIG. 37) while no change in GAPDH mRNA expression was observed in the RT-PCR reaction (FIG. 44).

To determine whether circular plasmids with any of the U6 promoters (pol III RNA) or CMV (pol II RNA) induce immune response at the cellular level, 5 constructs with any of the U6 or CMV promoters were used. Human glioma SNB19 cells were transfected with circular plasmids containing any of the

35 U6 or CMV promoters to direct the following: no insert called empty vector (EV), GFP RNAi insert that did not form perfect fork structure called encoded vector (SV), RNAi fork expression element for uPAR (puPAR) , RNAi hairpin expression element for uPA (puPA), a double RNAi hairpin expression for both uPAR and uPA (pU2). RT-PCR was performed to determine OAS1 expression levels. Total RNA was isolated from each of the transfected cells after 48 h of transfection and RT-PCR was performed to determine the level of OAS1 expression by 50 ng of total RNA. (The RT-PCR was performed according to the manufacturer's instructions (Invitrogen)). There was no change in OAS1 mRNA levels or GAPDH mRNA levels in SNB19 cells transfected with circular plasmids containing any of the U6 or CMV promoters (FIG. 38).

Example 32. Comparison of RNA pol II (CMV) and RNA pol III (U6) as promoters for the initiation of RNAi.

To determine the activity and efficacy of pol II RNA and pol III RNA, RNAi vectors were constructed in plasmid pSilenciador (Ambion, Austin TX) for encoded vector, uPAR, uPA and combination of uPAR-uPA as well as pcDNA3. The pSilenciador constructs were terminated with tetra Ts according to the manufacturer's instructions. SNB19 cells were transfected into two sets, one set contained polV RNA II CMV promoter and the second set contained pol III RNA U6 promoter (C, SV, puPAR, puPA and pU2). 48 h after transfection, the proteins were extracted from the cells according to conventional protocols and loaded on a 12% SDS polyacrylamide gel (10 µg / lane). Western blotting was performed and fibrin zymography was performed according to conventional protocols and investigated for uPAR and uPA and load control was determined.

55 through research for GAPDH. From FIG. 39, it was evident that the pol II RNA promoter constructs were more effective in the negative regulation of the target molecules when compared to the pol III RNA promoter constructs.

Example 33. Determination of the OAS1 gene for interferon response.

Plasmid constructs for empty vector (EV), encoded vector (SV), uPAR (puPAR), uPA (puPA), and the bicistronic construct for uPAR and uPA (pU2) were used to determine the level of interferon induction in the line of human glioma SNB19 cells. OAS1 gene expression was used as an indicator for interferon induction. Circular plasmids (C), linear expression cassette (L), and linear expression cassette were used as a consequence of suppressed BGH poly A signal (∆A). SNB 19 cells are transfected with equivalent amounts of the plasmid or expression cassettes mentioned above (schematic representation of

image21

C, L, ∆A) and total RNA was isolated after 48 h of transfection using conventional protocols. RT-PCR was performed on the samples mentioned above and OAS1 amplicon levels were determined on agarose gel. The primers used for amplification of OAS1 were 5’-aggtggtaaagggtggctcc-3 ’and 5’acaaccaggtcagcgtcagat-3’. The primers used to amplify the plasmid expression cassette

5 mentioned above (EV, SV, puPAR, puPA and pU2) were: direct primer 5’ctggtgtcgacctgcttccgcgatgtacgggc3 ’and reverse primer 5’ctggtgtcgacatccccagcatgcctgctat3’ (FIG. 40).

RT-PCR for induction of OAS1 mRNA (2.5'-oligoadenylate synthetase) was performed to determine the importance of a poly A signal sequence. Circular, linear (expression cassette only) and expression with sequence cassettes were used. suppressed poly A signal (C, L, and -A respectively). In the case of EV, the overall length of the transcript was not expected to be greater than 1 kb and the predicted structure of the transcript did not have a significant structure of RNAds to induce an immune response with or without a poly A tail as observed in the figure (also in SV) (FIG. 44). On the contrary, with puPAR, puPA and pU2, the predicted secondary structure had RNAd structures but with the presence of a poly A tail, although the

15 induction of immune response (expression of OAS1). In the case of expression cassette alone, when a poly A signal sequence was present but the transfected construct was linear, it induced an immune response. This indicated that the presence of a circular molecule produced a viable poly A tail; and since the linear construct was terminated just before the poly A signal sequence, the beginning of a viable poly A tail did not start or was incomplete. In the case of linear constructs with a suppressed poly A signal sequence, the immune response was initiated, indicating that the presence of a poly A tail may be important in the prevention of an immune response and in the stability of the molecule. of transcribed RNA (FIGS. 41-42).

The predicted mRNA of the bicistronic construct did not resemble that of mRNA and had a perfect loop and hairpin structure for both uPAR and uPA sequences. A sequence of 48 bases was introduced forming a

25 partial RNAs of 24 bases between the uPAR and uPA sequences to allow efficient transcription of both siRNA molecules. The bicistronic sequence was terminated with a poly A sequence encoded with a BGH poly-∆A signal sequence (FIG. 43).

RT-PCR for the OAS1 gene, a classic antiviral response gene, indicated that there was no immune response as did the transfected control cells and EV / SV. RT-PCR was also performed for transcripts of uPA and uPAR in antisense cells and transfected with RNAi. As determined with RT-PCR, no change was observed in uPAR or uPA mRNA transcripts in the antisense transfected cells, while uPAR or uPA mRNA levels in the RNAi transfected cells were reduced, which indicates a destruction of the respective mRNA (24 h). The mechanism of RNAi involves the destruction of target mRNA molecules. The

The expression of OAS1 was similar to that of the control groups (pGFP, pEV and pSV) indicating the absence of immune response at the cellular level (FIG. 44).

Example 34. In situ localization of RNAi expression vectors.

The paraffin embedded sections were deparaffinized and rehydrated according to conventional protocols under conditions without nuclease. These sections were treated with proteinase K to reveal any protein-bound DNA. The DNA was denatured according to conventional protocols. The pcDNA 3 plasmid was taken and the expression cassette containing the CMV promoter (digested with Nru I Hind III) was labeled with thermostable alkaline phosphatase (Amersham Biosciences, Piscataway, NJ) and hybridized with the treated sections. The

45 hybridization was performed according to the manufacturer's instructions. Mice injected with simulated had no alkaline phosphatase activity, while mice treated with IP injections of EV, SV, puPAR, puPA or pU2 had alkaline phosphatase activity, indicating the presence of the CMV promoter. Alkaline phosphatase activity was located most of the cases around the blood vessels and showed radiant patterns around the vasculature, indicating the crossing of the plasmid vectors that carry CMV through the blood brain barrier (FIG. 45 ).

Example 35. Determination of the OAS1 gene for interferon response for circular plasmids of uPAR-Catepsin B.

55 Plasmid constructs for empty vector (EV), encoded vector (SV), uPAR (pU), cathepsin B (pC), and the bicistronic construct for uPAR and cathepsin B (pCU) were used to determine the level of interferon induction in the human glioma SNB19 cell line. The genetic expression of OAS1 was used as an indicator of interferon induction. Circular plasmids (C), linear expression cassette (L), and linear expression cassette with suppressed BGH poly A signal sequence (-A) were used. SNB19 cells were transfected with equivalent amounts of the plasmid or expression cassettes mentioned above and the total RNA was isolated after 48 h of transfection using conventional protocols. RT PCR was performed on the aforementioned samples and OAS1 amplicon levels were determined on an agarose gel. The primers used for the amplification of OAS1 were 5’-aggtggtaaagggtggctcc-3 ’and 5’-acaaccaggtcagcgtcagat-3’. The primers used to amplify the expression cassette of the aforementioned plasmids (EV, SV, pU, pC and pCU) were:

65 direct primer 5’ctggtgtcgacctgcttccgcgatgtacgggc3 ’and reverse primer 5’ ctggtgtcgacatccccagcatgcctgctat3.

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RT-PCR for induction of OAS1 mRNA (2 ', 5'-oligoadenylate synthetase) was performed to determine the importance of a poly A signal sequence. Circular, linear cassettes (expression cassette only) and expression cassette were used. suppressed poly A signal sequence (C, L, and -A respectively). In the case of EV and SV, OAS1 mRNA induction was not detected. In the case of EV, the overall length of the transcript was not expected to be greater than 1 kb and the predicted structure of the transcript did not have a significant RNAd structure to induce an immune response with or without a poly A tail as observed. in the figure (also in SV). On the contrary, with pU, pC and pCU, the predicted secondary structure had RNAd structures but with the presence of a poly A tail, although the induction of immune response was not detected (expression of OAS1). In the case of expression cassette alone, when a poly A signal sequence was present but the transfected construct was linear, it induced an immune response. This indicated that the presence of a circular molecule produced a viable poly A tail; and since the linear construct was terminated just before the poly A signal sequence, the beginning of a viable poly A tail did not start or was incomplete. In the case of linear constructs with a suppressed poly A signal sequence, the immune response was initiated, indicating that the presence of a poly A tail may be important in the prevention of an immune response and in the stability of the molecule. of RNA

15 transcribed.

The induction of OAS1 was examined in intracranial xenograft tumors treated with EV, SV, pU, pC and pCU. Normal mice that were treated with interferon α (3 µg / mouse) intracranially were included and sacrificed 5 hours later. The spleen and liver were used as normal control tissues to corroborate the specificity of the antibody in which the presence of OAS1 expression will be present under normal conditions. In addition, from immunohistochemistry, in situ hybridization was performed on these tissues using sense oligos (acaaccaggtcagcgtcagat) to determine RNA levels in OAS1. There was only a very minimal expression of OAS1 mRNA and protein in mouse brains with intracranial tumors with intracranial or in the brains of mice treated with pU, pC and pCU. In particular, no induction of OAS1 occurred in the group treated with pCU.

25

MATERIALS AND METHODS

Plasmid construction expresses small hairpin RNA:

uPA-uPAR: A small interfering oligonucleotides specific for uPA from 346 to 367 bases (agcttGagagccctgctggcgcgccatatataatggcgcgccagcagggctctca) and for uPAR from 77 to 98 bases (gatccTacagcagtgtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtagtag. A uPAuPAR RNAi plasmid vector that expresses the shRNAs for both uPA and uPAR was constructed under the control of a human CMV promoter by inserting pairs of hybridized DNA oligonucleotides specific for uPA at the Hind III site and

35 uPAR at the BamHI site sequentially in the pcDNA3 vector (sh-uPAuPAR). RNAsh expression vectors were also constructed for uPA (sh-uPA) and uPAR (sh-uPAR) individually. A vector encoded with pcDNA3 with an imperfect sequence, which is not part of the perfect fork structure, was used to develop the vector encoded for use as a control. The empty vector (EV) and encoded vector (SV) controls have been tested on multiple cell lines and do not demonstrate any toxicity with respect to cells as demonstrated by the MTT test after transfection as well as having no effect on the expression of constitutive genes, GAPDH and γ-actin.

uPAR and MMP-9. PcDNA 3 was used to construct a vector that expresses siRNA for both uPAR and MMPs-9 downstream of the cytomegalovirus (CMV) promoter (Scheme 1). The uPAR sequence from +77 to +98

45 was used as the target sequence and for convenience a oligo of self-complementarity was used. The 21 base length uPAR sequence with a 9 base loop region and BamHI sites were incorporated at the ends (gatcctacagcagtggagagcgattatatataataatcgctctccactgctgtag). The oligo was autohybridized in 6x SSC using conventional protocols and ligated into the BamHI site of a plasmid vector of pcDNA-3. Similarly, a complementary MMP-9 sequence from +360 to +381 (aattcaagtggcaccaccacaacaatatataattgttgtggtggtgccacttg) with EcoRI sites incorporated at the ends was ligated into the EcoRI site of the vector containing the siRNA sequence for uPAR. Finally, this resulted in an siRNA expression plasmid for uPAR and MMP-9 with a separation of 35 bp. The orientation of any insert did not matter since the oligos are self-complementary and have a bilateral symmetry. The SV40 terminator served as a stop signal for RNA synthesis.

55 Cathepsin B and uPA: pcDNA 3 was used to construct a vector that expresses siRNA for both cathepsin B and for uPAR downstream of the cytomegalovirus (CMV) promoter (FIG. 17). The uPAR sequence from +77 to +98 was used as the target sequence and for convenience a self-complementary oligo was used. The 21 base length uPAR sequence was used with a 9 base loop region with BamHI sites incorporated at the ends (gatcctacagcagtggagagcgattatatataataatcgctctccactgctgtag). The oligo was autohybridized in 6x SSC using conventional protocols and ligated into the BamHI site of a plasmid vector of pcDNA-3. Similarly, a complementary sequence of cathepsin B from +732 to +753 (tcgaggtggcctctatgaatcccaatatataattgggattcatagaggccacc) with XhoI sites incorporated at the ends was ligated into the XhoI site of the vector containing the siRNA sequence for uPAR. Finally, this resulted in an siRNA expression plasmid for cathepsin B and uPAR called pCU. Individual siRNA expression vectors were also constructed for uPAR (pU) and

65 cathepsin B (pC). The orientation of any insert in the individual or bicistronic did not matter since the oligos were self-complementary and presented bilateral symmetry. A BGH poly A terminator served as a signal of

stop for RNA synthesis of the three constructs.

uPAR, uPA and MMP-9: pcDNA3 was used to construct a vector that expresses siRNA for uPAR, uPA and MMP-9 downstream of the cytomegalovirus (CMV) promoter. The uPAR sequence from +77 to +98 was used as the target sequence and for convenience a self-complementary oligo was used. The 21 base length uPAR sequence was used with a 9 base loop region with BamHI sites incorporated at the ends (gatcctacagcagtggagagcgattatatataataatcgctctccactgctgtag). The oligo was autohybridized in 6x SSC using conventional protocols and ligated into the BamHI site of a plasmid vector of pDNA3. Similarly, the complementary sequence of +346 to +367 uPA (agcttgagagccctgctggcgcgccatatataatggcgcgccagcagggctctca) with HindIII sites incorporated at the ends was ligated into the HindIII site and MMP-9 +360 to +381 (aattcaagtggcaccaccacaacaatatataattgttgtggtggtgccacttg) was ligated into the EcoRI site of the vector containing the siRNA sequence for uPAR and uPA. Finally, this resulted in an siRNA expression plasmid for uPAR, uPA and MMP-9 called pU2M. Individual siRNA expression vectors were also constructed for uPAR (puPAR), uPA (puPA) and MMP-9 (pMMP-9). The orientation of the insert in any of the construct

15 individual or tricistronic was not a factor because the oligos were self-complementary and presented bilateral symmetry. The BGH poly A terminator served as a stop signal for RNA synthesis for the four constructs.

Cathepsin B and MMP-9: Autocomplementary inverted repeat sequences spaced with a 9-base G and C deficient region directed to cathepsin B (732-753) and MMP-9 (360-381) were synthesized. The oligos for cathepsin B were terminated with XhoI sites and the oligos for MMP-9 were terminated with EcoRI and self-hybridized by heating at 100 ° C for 5 min and cooled to room temperature in 6x SSC which would result in the formation of molecules of double stranded DNA with the respective ends of adhesive detention sites. These dsDNA molecules bound to the XhoI and EcoRI sites of the plasmid vector of pCDNA, giving

25 resulted in the formation of a plasmid containing inverted repeats for cathepsin B and MMP-9 downstream of the CMV promoter and terminated with an SV40 terminator. The resulting plasmid called pCM transfected into mammalian cells would result in the production of a double hairpin siRNA molecule targeting both Cathepsin B and MMP-9 that would be further processed by an enzymatic recognition mRNA (DICER) to produce molecules of Individual siRNAs to induce RNAi (Scheme 1).

Culture conditions and cell transfection:

Prostate cancer cells: Human prostate cancer cell lines, LNCaP, DU145 and PC3, were obtained in the American Type Culture Collection (Manassas, VA). LNCaP cells were grown in RPMI medium

35 supplemented with 2 mM L-glutamine, 1.5 g / l sodium bicarbonate, 4.5 g / l glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate (Invitrogen, Carlsbad, CA). PC3 and DU145 cells were cultured in minimal essential medium. Both media contained 10% fetal bovine serum (GIBCO BRL, Lewisville, TX) and 5% penicillin / streptomycin and were kept in an incubator at 37 ° C in a humidified atmosphere with 5% CO2. Transfections were performed using the Lipofectamine ™ 2000 reagent (Life technologies, Rockville, MD) according to the manufacturer's instructions. After 72 h of transfection, the cells used for cell proliferation assays, immunoblot analysis, RT-PCR analysis, Matrigel invasion assay, DNA fragmentation assay, EMSA assay and caspase activity assay. For immunostaining with DAPI and double, transfections were performed on Lab-Tek II camera slides (Nalge Nunc International, Naperville, IL).

45 Glioblastoma cells: The human glioblastoma SNB19 cell line maintained in DMEM F-12 (Sigma Chemical Co., St. Louis, MO) supplemented with 10% FCS, 100 pg / ml streptomycin and 100 units / ml of penicillin (Invitrogen, Carlsbad, CA) at 37 ° C in a humidified 5% CO2 atmosphere. Cells were transfected with pC pU or pCU plasmid expressing siRNA using the Lipofectamine reagent (Invitrogen Grand Island, NY) according to the manufacturer's instructions. After transfection, the cells were incubated in medium containing serum for 48 h.

Fibrin zymography: Enzymatic activity and molecular weight of electrophoretically separated forms of uPA were determined in conditioned media of prostate cancer cell lines, LNCaP, DU145 and PC3, with SDS-PAGE. In summary, the SDS-PAGE gel contains acrylamide to which they were added.

55 purified strains of plasminogen and fibrinogen before polymerization. After polymerization, equal amounts of proteins in the samples were electrophoresed in the gel that was washed and stained. SNB 19 cells transfected with EV / SV puPAR, puPA, pMMP-9 and that pU2M were also prepared as described herein.

Jello Zymography Conditioned media were collected from transfected with EV / SV, puPAR, pMMP-9 and pUM and centrifuged to remove cell debris. Twenty micrograms of the resulting samples were tested for gelatinase activity using 10% sodium dodecyl sulfate-polyacrylamide angels containing gelatin (0.5 mg / ml). The gels were stained with Amido black (Sigma Aldrich ST LOUIS MO) and the gelatinase activity was visualized in the form of areas with transparent bands on the gels. SNB 19 cells

Transfected with EV / SV, puPAR, puPA, pMMP-9 and pU2M were also prepared as described herein.

image23

Reverse transcription analysis-PCR:

uPA-uPAR: Cellular RNA was isolated using the QNegen RNeasy kit and 1 g of RNA was treated with DNase (10 units / g of RNA, 1 h) and used as a template for the reverse transcription reaction (RT, 20 one). The 5 RT reaction mixture (Invitrogen) contained 11 (10 pm) of primers. The resulting cDNA was then used in PCR reactions and analyzed by gel electrophoresis. The following primers were used:

uPA-sense: 5’TGCGTCCTGGTCGTGAGCGA 3 ’; uPA-antisense: 5’CTACAGCGCTGACACGCTTG 3 ’;

10 uPAR-sense: 5’CATGCAGTGTAAGACCCAACGGGGA 3 ’; uPAR-antisense: 5’AATAGGTGACAGCCCGGCCAGAGT 3 ’; GAPDH-sense: 5’CGGAGTCAACGGATTTGGTCGTAT 3 ’; and GAPDH-antisense: 5’AGCCTTCTCCATGGTGGTGAAGAC3 ’.

15 Table: 1

RT-PCR primers (MMP-9, UPAR)

uPAR CATGCAGTGTAAGACCCAACGGGGA

AATAGGTGACAGCCCGGCCAGAGT

MMP-9

GTTCGAAATTAGTTTGGTTAAC

CCGAATAACTAATATTATAAACG

GAPDH

CGGAGTCAACGGATTTGGTCGTAT

AGCCTTCTCCATGGTGGTGAAGAC

Used probes

sGFP (3) GAGCTGTTCACCGGGGTGGTG

suPAR (1)

CTACAGCAGTGGAGAGCGATT

sMMP-9 (2)

CAAGTGGCACCACCACAACAA

RT-PCR analysis for SNB19 cells transfected with control / EV, SV, puPAR, pMMP-9 and pUM was performed as described herein.

20 The PCR conditions were as follows: 95 ° C for 5 minutes, followed by 35 titers of 95 ° C for 1 min, 55 ° C for 1 minute, and 72 ° C for 1 minute. The final extension occurred at 72 ° C for 5 min. The hybridization temperature varies depending on the sequence of the various constructs and was performed following conventional procedures.

25 PC3 Immunofluorescence Detection: PC3 cells transfected with various plasmids of shRNA with 4% paraformaldehyde were fixed and incubated with anti-uPA (1: 500; Biomeda, Foster City, CA) and / or anti-uPAR

(1: 500; American Diagnostics Inc., Greenwich, CT). After washing, fluorescent secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were added with a dilution to 1: 500. The cells were washed again three times with PBS, stained with DAPI. Fluorescence images were taken using an RT Camera

30 Slider Spot with attached charging device (Diagnostic Instruments Inc, Burroughs Sterling Heights, MI) connected to a microscope (Olympus, Melville, NY) and directed with a computer equipped with the v3.5 RT software for point application (Diagnostic instruments, Burroughs Sterling Heights, MI).

PC3 Cell Invasion Assay: After transfection, the cells were separated and washed twice

35 in PBS. 5x105 cells were seeded in the upper chamber of a Transwell insert (12 µM pores) coated with Matrigel (0.7 mg / ml) (Collaborative Research Inc., Boston, MA). The lower chamber was filled with 4001 RPMI medium. After an incubation period of 24 h, the non-migrated cells in the upper chamber were carefully scraped off and the adherent cells present on the lower surface of the insert were stained with Hema-3 and photographed.

40 On-site caspase activity assay: Caspase activation was detected using the policaspase detection kit (Immunochemistry Technologies, Bloomington, IL) according to the manufacturer's instructions. In this assay, non-cytotoxic, permeable fluorochrome Caspase inhibitors (FLICA) bind covalently to a reactive cysteine residue in the large subunit of the active caspase heterodimer,

45 thereby further inhibiting enzymatic activity. This kit uses a peptide inhibitor of many carboxy fluororescein-labeled fluoromethyl ketone caspases (caspase 1, -3, -4, -5, -6, -7, -8 and -9; FAM-VAD-FMK), which is a Generic probe for the detection of most caspases and emits green fluorescence. The green fluorescent signal is a direct measure of the amount of active caspase in the cell at the time the reagent is added. After 72 h of transfection, caspase activation was detected

50 by staining the cells with the FAM-VAD-FMK dye (in situ marker). The bound marker was located by fluorescence detection as observed with a confocal microscope. For nuclear staining, DAPI was used.

image24

DNA marker assay:

5 and 1% Triton X-100) containing 0.1 mg / ml Proteinase K (Invitrogen) and then incubated at 37 ° C for 2 h. The DNA was removed from the lysates by centrifugation and then extracted using an equal volume of phenol / chloroform and precipitated by the addition of absolute ethanol and 0.3 M sodium acetate (pH 5.2) at -80 ° C for 2 h . The DNA was resuspended in Tris-EDTA buffer (10 mM Tris-HCl; pH 7.5, 1 mM EDTA), treated with RNase A at 37 ° C for 1 h, and then resolved on an agarose gel at 1.5% stained with ethidium bromide (0.5 g / ml).

Electrophoretic mobility shift test (EMSA): After transfection, nuclear proteins were extracted using a protein extraction kit (Ambion, Austin, TX) according to the manufacturer's instructions. The concentrations of nuclear proteins were determined in our dilutions using a

15 procedure with bicinconic acid (Pierce Biochemical Company, Rockford, IL). The interaction between Stat 3 in the protein stratum and the DNA probe was investigated using an electrophoretic mobility shift test kit (EMSA) from Panomics (Redwood City, CA) according to the manufacturer's instructions.

DNA fragment end labeling assay: sections of prostate tumor tissue treated with shRNA or control (5 M thick) were deparaffinized and rehydrated. Next, the tissue sections were permeabilized and covering the entire test sample with Proteinase K solution (20 g / ml Proteinase K in 10 mM Tris, pH 8) and incubated for 20 min at room temperature. Next, the tissue sections were washed with Tris buffered saline (1x of TBS, 20 mM Tris at pH 7.6, 140 mM NaCl). The inactivation of the endogenous peroxidases was achieved by immersing the tissue sections in 3% hydrogen peroxide diluted in methanol for 5 min at room temperature. The glass slides were then placed in Klenow equilibrium buffer (50 mM Tris at pH 8, 50 mM NaCl, 10 mM MgCl 2) for 30 min. Next, the tissue sections were incubated with 60 µl of a solution containing a mixture of labeled and unlabeled deoxynucleotides at an optimal rate of marking the end of the DNA fragment with Klenow, according to the manufacturer's instructions (kit Klenow-FragEL DNA fragmentation detection, Oncogene Research Products, Cambridge, MA) at 37 ° C for 90 min in a humidified chamber. The enzymatic reaction was stopped by incubation with EDTA (0.5 M, pH 8) for 5 min at room temperature. The slides were then washed with TBS and immersed in blocking buffer for 10 min (4% BSA in PBS) followed by incubation with 100 µl of a solution containing streptavidin peroxidase for 30 min in a humidified chamber at room temperature. Then the sections of

The tissue was washed in TBS and covered with a solution containing 3, 3 'diaminobenzidine (DAB, 0.7 mg / ml), hydrogen peroxide and urea (0.6 mg / ml). Then, the slides were washed with distilled water and counterstained with methyl green (0.3%) for 30 seconds and examined in an Olympus fluorescence microscope. Marked positive staining of the end of the DNA fragment was scored from six images / samples captured randomly using the v3.5 RT application software (Diagnostic instruments, MI).

Orthotopic mouse prostate treatment model: Nude male nude mice (nu / nu; 6-8 weeks of age) were obtained at Harlan Sprague-Dawley (Indianapolis, IN). Animal handling and experimental procedures are approved by the animal experiments committee of the University of Illinois 45 College of Medicine. Orthotopic implantation was performed as described above. In summary, after total anesthesia of the body with ketamine (50 mg / kg) and xylazine (10 mg / kg), an incision was made in the lower midline in the lower abdomen. A suspension of PC3 cells (1 x 106) in 30 µl of PBS was injected into a lateral lobe of the prostate and the wound was closed with surgical metal clips. This cell concentration was necessary to achieve consistent local tumor growth 7 days after implantation. The mice were divided into five treatment groups with six mice per treatment group. On days 7 and 14 after implantation, a midline low incision was made and the tumors were injected with plasmid constructs expressing sh-uPA, sh-uPAR, sh-uPA-uPAR or EV / SV controls ( 75 µg / 150 µg each). In another set of experiments, the orthotopically implanted mice were co-injected intratumorally with plasmids of sh-uPA and sh-uPAR (150 µg each) on days 7 and 14. The mice were sacrificed 14-15 days after injection

Final plasmid mRNA and primary tumor growth and metastasis sites were determined by visual inspection and photographed. Next, the primary tumors were excised, measured and weighed. The test samples were fixed in formalin and embedded in paraffin for staining with H and E. In addition, a part of the tissue was instantly frozen immediately for immunoblotting.

Western transfer. SNB19 cells were transfected with simulated, empty vector, pC, pU or pCU and cultured 48 h. At the end of the incubation, the cells were harvested, washed twice with cold PBS and lysed in buffer (150 mM NaCl, 50 mM Tris-HCl, 2 mM EDTA, 1% NP-40, pH 7.4) , which contained protease inhibitors. Equal amounts of protein (30 µg / lane) from supernatants or cells were electrophoresed under non-reducing conditions in 10% acrylamide gels. After SDS-PAGE, proteins 65 were transferred to a polyvinylidene difluoride membrane (Bio-Rad). To block non-specific binding, the membrane was incubated for 2 h in PBS with 0.1% Tween-20 [T-PBS] containing 5% fat-free skim milk for 2 h. Subsequently, the membrane was incubated for 2 h with antibody against cathepsin B, uPAR, ERK, pERK, FAK or pFAK respectively in T-PBS + 5% fat-free milk. After washing in T-PBS, the membrane protein was visualized using the ECL ™ detection kit with a peroxidase-labeled anti-rabbit antibody (Amersham Pharmacia Biotech, Amersham, UK) according to the manufacturer's instructions. For load control, the membranes were separated and investigated with monoclonal antibodies to β-actin, according to conventional protocols. Immunostaining analysis for SNB19 cells that were transfected with EV / SV, puPAR, puPA, pMMP-9 and pU2M was also performed as described herein. The following antibodies were used for the immunoblot analysis of uPA-uPAR: anti-uPA (Biomeda, Foster City, CA), anti-uPAR (American Diagnostics Inc., Greenwich, CT), anti-Bax (Santa Cruz Biotechnology, Santa Cross,

image25

10 CA), anti-Bcl-XS / L, (Santa Cruz Biotechnology, Santa Cruz, CA), anti-caspase 9 (Cell Signaling Technology Inc., Beverly, MA), and anti-GAPDH (Abcam, Cambridge, MA) . Antibodies to the total and phosphorus forms of ERK, JNK, p38 and Stat 3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Western blotting for SNB19 cells that were transfected with EV / SV, puPAR, puPA, pMMP-9 and pU2M was also performed as described herein.

15 Immunohistochemical analysis. SNB19 cells (1 x 104) were seeded in 8-well chamber slides coated with vitronectin, incubated for 24 h and transfected with EV / SV, puPAR, pMMP-9 and pUM. After another 72 h, the cells were fixed with 3.7% formaldehyde and incubated with 1% bovine serum albumin in PBS at room temperature for 1 h for blocking. Then the slides were washed with

20 PBS, either anti-uPAR IgG (rabbit) or anti-MMP-9 IgG (mouse) was added at a concentration of 1: 200. The slides were incubated at 4 ° C overnight and washed three times with PBS to remove excess primary antibody. The cells were then incubated with anti-mouse FITC conjugate IgG or anti-FITC conjugates (1: 500 dilution) for 1 h at room temperature. The slides were then washed three times, covered with glass coverslips and fluorescent photomicrographs were obtained. They were obtained

25 composite mixed images to visualize the expression of uPAR and MMP-9 in cells transfected with control / EV, SV, puPAR, pMMP-9 and pUM.

SNB19 Cell Proliferation Assay. Cell growth was assessed by MTS assay. To detect the effect of these constructs on the growth of SNB19 cells in vitro, the mass of viable cells was measured using the Cell Titer 96 ™ colorimetry assay. 5 x 003 glioblastoma cells were seeded in triplicate in 96 or 24 well plates and allowed to grow for 24 h before transfection with culture medium vectors only (simulated), EV, SV, pC, pU and pCU during 48 h These cells were then changed to medium containing serum and different time intervals were allowed. Before each time point, the MTS reagent was added and incubation continued for an additional 2 h period to allow color development.

35 A490 was measured in each well using an ELISA plate reader. Absorbance readings for short-term cell cultures with respect to long-term were compared, and the effects of these constructs were interpreted with respect to the growth of the corresponding untreated / control groups. The percentage of growth inhibition due to the siRNA construct was calculated with respect to the growth rate of the same cells in the same medium minus these constructs.

40 PC3 cell proliferation assays: Cell viability 72 h after transfection was evaluated using an MTT assay. MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] (Sigma) was added to the culture medium in each well at a concentration of 500 g / ml, and the plates were incubated for 4 h at 37 ° C. Isopropanol acid (0.04 N HCl / isopropanol) was immediately added to the wells and vigorously mixed with

45 so that the dark blue crystals dissolved efficiently. The absorbance was measured 570 nm (Benchmark, BIORAD, Hercules, CA).

In vitro angiogenic test of SNB19. SNB19 cells (2 x 104) were seeded on 8-well chamber slides and transfected with simulated, EV, pU, pC and pCU according to conventional protocols. After 50 of a 24 h incubation period, the medium was removed and 4 x 104 human dermis endothelial cells were seeded and coculturing was allowed for 72 h. After fixation in 3.7% formaldehyde, endothelial cells were immunologically investigated for antigenic factor VIII. Factor VIII antibody was purchased from DAKO Corporation (Carpinteria, CA). The cells were washed with PBS and incubated with FITC conjugated to secondary antibody for 1 h. and then washed and examined with a fluorescence microscope. Similar slides of endothelial cells cultured in the presence of conditioned media from the SNB 19 cells transfected with the simulated, EV, pU, pC or pCU were stained with H and E to visualize the formation of networks. For the quantification of angiogenesis, the Image Pro soft software was used, the degree of angiogenesis was measured with the following method: the number of branch points and the total number of branches per point were randomly counted (by 10 fields) , with the product indicating the grade

60 angiogenesis compared to controls. In vitro angiogenic assays for SNB19 cells (2 x 104) seeded on 8-well chamber slides and transfected with simulated / EV, puPAR, pMMP-9 and pUM were performed as described herein. Angiogenic assays for SNB19 cells (1 x 104 well-1) seeded on 8-well chamber slides, incubated for 24 hours and transfected with EV / SV, puPAR, puPA, pMMP-9 and which pU2M were performed as described in This document.

65

image26

Dorsal skin fold chamber model: Alchemical nude mice (nu / nu; 18 male / female, 28-32 g) were fed and maintained within a germ-free environment with specific pathogens with the technique of implanting the chamber model. dorsal skin fold. Sterile surgical techniques were followed for small animals. Mice anesthetized by ip injection with ketamine (50 mg / kg) and zylazine 5 (10 mg / kg). Once the animal was fully anesthetized, a dorsal air sac was made in the mouse by injection of 10 ml of air. Diffusion chambers (Fisher) were prepared by aligning 0.45 micrometer membranes of Millipore (Fisher) on both sides of the edge of the "O" ring (Fisher) with sealing. Once the chambers were dry (2-3 min), they were sterilized with UV variation for 20 min. 20 µl of PBS was used to moisten the membranes. 2 x 106 SNB 19 cells were injected (simulated, empty vector

or transfected pCU), suspended in 100-150 µl of sterile PBS, in the chamber through the "O" ring opening. The opening was sealed with a small amount of bone wax. A superficial incision of ½ to 2 cm was made horizontally along the edge of the dorsal air sac and the air sac that opened. With the help of forceps, the cameras were placed under the skin and carefully sutured. After 10 days, the animals were anesthetized with ketamine / xylazine and sacrificed by intracardiac infusion with solution

Saline (10 ml) followed by a 10 ml solution of 10% formalin / 0.1 M phosphate and followed by 0.001% FITC solution in PBS. The animals were carefully sensitized around the implanted chambers and the implanted chambers were removed from the s.c. The skin fold covering the cameras was photographed with visible light and for FITC fluorescence. The numbers of blood vessels within the chamber in the fascia area of the air sack were counted and their lengths measured. SNB 19 cells transfected with EV / SV, puPAR, pMMP-9 and pUM were also used for dorsal skin fold chamber model as described herein.

SNB19 Cell Migration Assay: A 2 x 106 suspension in Dulbecco's modified Eagle's medium of a variant of GFP expression of SNB19 cells was pipetted into 100 mm ultra low-junction 100 mm tissue culture plates and grown to spheroid aggregates formed. Spheroids that measured ~ 150 µm in diameter (approximately 4 x 104 cells / spheroid) were selected, transfected with simulated, empty vector, pC, pU and pCU and cultured for 48 h. 72 h after transfection, a single glioma spheroid was placed in each well of a 96-well microplate coated with vitronectin (50 µg / ml) and cultured with 200 µl of free medium without serum. The spheroids were incubated at 37 ° C for 24 h, after which the spheroids were fixed and stained with Hema-3 and photographed. Cell migration from spheroid to monolayers was measured using a microscope calibrated with an ocular micrometer and platform and used as an index of cell migration. Glioblastoma cells were seeded in triplicate in 96 or 24-well plates and allowed to grow for 24 h before transfection with culture medium alone (simulated), EV / SV, puPAR, pMMP-9 and pUM as described in the present document. Cell migration assays were performed for SNB19 35 cells transfected with EV / SV, puPAR, pMMP-9 and pUM as described herein. Assays for SNB19 cells (1 x 104 well-1) seeded on 8-well chamber slides, incubated for 24 hours and transfected with EV / SV, puPAR, puPA, pMMP-9 and pU2M were performed as described herein. document.

Boyden chamber invasion assay of SNB19 cells: The ability of in vitro invasion of SNB19 cells in the presence of the siRNA expressing vector for cathepsin B and uPAR was evaluated using a modified Boyden chamber assay. SNB19 cells were transfected with simulated, EV, pU, pC or pCU vector expressing siRNA for cathepsin B and uPAR individually or together for 48 h. 1 x 106 cells were suspended in 600 µl of media and serum supplemented with 0.2% BSA and placed in the upper compartment of the Transwell chambers (Cat. No. 07-200-158 of Corning Costar Fischer Scientific, Pittsburg, PA ) coated with Matrigel (0.7

45 mg / ml) The lower chamber compartment was filled with 200 ml of serum-free medium and the cells were allowed to migrate for 24 h. After incubation, the cells were fixed and stained with Hema-3 and photographed. Invasion assay quantification was performed. Assays for SNB19 cells (1 x 104 well-1) seeded on 8-well chamber slides, incubated for 24 hours and transfected with EV / SV, puPAR, puPA, pMMP-9 and pU2M were performed as described herein. document.

Spheroid SNB19 Assay: SNB19 glioblastoma cells (3 x 106) were seeded in 100 mm tissue culture plates (Coming, Corning, NY) you will pre-coat with 0.75% agar prepared in DMEM and cultured until they were they formed spheroid aggregates. 100-200 µm diameter spheroids were selected and transfected with simulated, empty vector, pC, pU and pCU for 48 h. Three days after infection, SNB19 spheroids were stained with DiI fluorescent dye and placed in contact with fetal rat brain aggregates stained with DiO. The progressive destruction of fetal rat brain aggregates and invasion of SNB 19 cells were observed with confocal laser scanning microscopy and photographed as described above. The remaining volume of brain aggregates or tumor spheroids during cocultures was determined as described above. Spheroid assays for SNB19 cells transfected with EV / SV, puPAR, pMMP-9 and pUM were performed as described herein. Assays for SNB19 cells (1 x 104 well-1) seeded on 8-well chamber slides, incubated for 24 hours and transfected with EV / SV, puPAR, puPA, pMMP-9 and pU2M were performed as described herein. .

Experiments with mice for glioma analysis. SNB19 GFP cells (2 x 106) were injected into the brains

65 of athymic mice using a stereotactic framework. After 8-10 days, the mice were treated with simulated, empty vector of EV / SV, pC, pU and pCU. Intracranial administration of vectors in vivo was performed using pumps

Alzet mini-osmotic (Direct Corp. Cupertion, CA) at the rate of 0.25 µl / hr, simulated (PBS) of 150 µg of vector DNA, 150 µg of pC, 150 µg of pU, 150 µg of pCU or puPAR vector (150 µg), pMMP-9 vector (150 µg) and pUM vector (150 µg) were injected into the brain (100 ul per mouse). All experiments were performed in accordance with the institutional guidelines established by the Institutional Animal Control Users Committee that

5 approves the experiments at the University of Illinois College of Medicine in Peoria. After 5 weeks, or when the control mice began to show symptoms, the mice were sacrificed by cardiac perfusion with formaldehyde. The brains were then removed and embedded in paraffin according to conventional protocols. Sections were prepared and observed for GFP expression or stained with H and E. Sections were screened and acted semi-quantitatively for tumor size in each case. The average tumor area per section was used to calculate tumor volume and was compared between control and treated groups. Experiments were performed for SNB19 cells (1 x 104 well-1) that were seeded on 8-well chamber slides, incubated for 24 hours and transfected with EV / SV, puPAR, puPA, pMMP-9 and pU2M as described in the present document.

15 Statistical Analysis: Statistical comparisons were made using ANOVA for significance analysis between different values using GraphPad Prism software (San Diego, CA). The values are expressed as mean DT from at least three separate experiments and the differences were considered significant at a P value of less than 0.05.

Inhibition of intracranial tumor growth. For the intracerebral tumor model, 2 x 106 SNB19 GFP cells were injected intracerebrally into nude mice. The tumors were allowed to grow for 10 days. At this time, the animals were randomly classified into seven groups and 150 µg of each EV / SV, puPAR, puPA, pMMP-9 and pU2M construct were injected into the brain using Alzet mini pumps at the rate of 0.25 µl h -1 (six mice in each group). Five weeks after tumor inoculation, six mice were sacrificed.

25 each group by cardiac perfusion with 3.5% formaldehyde in PBS. Their brains were removed and placed in 4% paraformaldehyde for 24 hours, embedded in paraffin and sectioned. Sections were systematically identified for fluorescence with GFP to examine tumor growth under a fluorescence microscope. Sections were reviewed with concealment and scored semi-quantitatively for tumor size. The mean area of the tumor in each section was used to calculate the tumor volume and for comparison between control and treated groups.

Invasion test with Matrigel. The invasion capacity of the transfected SNB 19 cells was subjected to in vitro assays with the Boyden chamber invasion assay after transfection with any of the empty vector (EV) r the siRNA expressing vector for cathepsin B and MMP-9 ( pCM). In short, inserts were coated

35 Transwell with 8 µm pores with Matrigel (0.7 mg / ml) (Collaborative Research, Inc., Boston, MA). SNB19 cells were trypsinized and 500 µl of the cell suspension (1 x 10 6 cells / ml) was added to the wells in triplicate. After incubation for 24 h at 37 ° C, the cells that passed through the filters in the lower wells were quantified and expressed as a percentage of the sum of the cells in the upper and lower wells. The cells on the lower side of the membrane were fixed, stained with Hema-3 and photographed. Tests for other constructs described were also performed following the procedures described herein.

Administration of nucleic acids: The administration of nucleic acids is achieved using any type of method such as for example lipophilic agent, viruses including adeno, adeno associated or lenti or with virus

45 modified. Naked plasmid constructs that are circular or linear with blunt or adhesive ends, such as double stranded RNA, single stranded RNA, or DNA-RNA hybrids in which one of the strands is DNA and the other is RNA, are also used. main chain of ribose or deoxyribose in the same female. DNA, RNA or DNA-RNA hybrids coated with proteins or carbohydrates or combinations thereof, or used in conjunction with hormones or hormone derivatives. As therapeutic molecules, chemically modified DNA or RNA or DNA-RNA hybrids can also be used to induce the RNAi direction of uPAR, uPA, MMP-9 and Cathepsin B in any combination. The intended use would be made in eukaryotic organisms or cell lines, preferably in human and human cell lines.

Some therapeutic methods and compositions known to those skilled in the art for administering siRNAs

55 or the shRNAs are within the scope of this disclosure. For example, siRNAs, siRNAs and other nucleic acids disclosed herein can be administered in mammalian cells through techniques such as viral vector administration, lipofection, electrochemical and biochemical methods. Virus-based administration includes the generation of adenoviral, lentiviral, retroviral recombinant vectors or any suitable vector that houses a nucleic acid of interest and the administration of viral vectors using techniques known to those skilled in the art. Some liposome-based administration systems include lipofection and cardiolipin-based compositions. The direct administration of naked nucleic acids in combination with chemical or biochemical adjuvants is within the scope of the present disclosure. For example, some circular plasmids that house siRNAs against a target sequence such as, for example, uPA, uPAR, MMP-9, and cathepsin B can be injected directly by beautiful minister via route

65 intratumoral or can be injected intraperitoneally. Similarly, some synthetic or chemically prepared nucleic acids can also be administered intratumorally or intraperitoneally. In addition, selective or specific administration of siRNAs or nucleic acids expressing siRNAs can also be achieved through proper coupling with another agent such as a peptide nucleic acid (PNA) or an antibody or any suitable targeting agent. The techniques and methods mentioned above are suitable and can be adapted to administer nucleic acid sequences intercellularly, via

image27

5 intracellular, in vitro cell cultures, in vivo, within an organ, through the blood brain barrier, to prostate cancers, gliomas, breast cancers and colon cancers.

Sequences: Some are disclosed in this document for various siRNA constructs (partial sequence). The underlined part indicates the autocomplementary inverted repetitions. The unpublished letter indicates the sequence of 10 intervention loop.

UPAR-uPA

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UPAR-MMP-9

image29

20 UPAR CB

image30

image31

MMP9-CB

image32

UPAR, uPA and Cat B

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MENTIONED DOCUMENTS

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A multicistronic short interference RNA construct is disclosed comprising at least a first and second autocomplementary sequences, in which the construct is used to inhibit tumor formation and to reverse previously formed tumors.

The multicistronic construct mentioned above is also disclosed, in which the first autocomplementary sequence comprises a nucleotide sequence of the human urokinase plasminogen activator receptor (uPAR) and its complement, and the second autocomplementary sequence comprises a

25 nucleotide sequence of human urokinase plasminogen activator (uPA) and its complement.

The multicistronic construct mentioned above is also disclosed, in which the autocomplementary sequence of uPA is TGAGAGCCCTGCTGGCGCGCC-loop-GGCGCGCCAGCAGGGCTCTCA and the autocomplementary sequence of uPAR is CTACAGCAGTGGAGAGCGATT-loop-AATCGCTCTAG.

The multicistronic construct mentioned above is also disclosed, in which the autocomplementary sequences of uPAR and uPA are separated by an intervention sequence with a length of approximately 22-35 base pairs.

In addition, the multicistronic construct mentioned above is disclosed, in which the intervention sequence is AGCTTGGTACCGAG CTCG GATC.

The multicistronic short interference RNA construct mentioned above comprising at least a first and second autocomplementary sequences is also disclosed, in which the construct is used to inhibit tumor formation and to reverse previously formed tumors, and in which the The first autocomplementary sequence comprises a nucleotide sequence of the urokinase plasminogen activator receptor (uPAR) and its complement and the second autocomplementary sequence comprises a nucleotide sequence of type 9 matrix metalloprotease (MMP-9) and its complement.

The multicistronic construct mentioned above is also disclosed, in which the autocomplementary sequence of uPAR is CTACAGCAGTGGAGAGCGATT-loop-AATCGCTCTCCACTGCTGTAG and the autocomplementary sequence of MMP-9 is CAAGTGGCACCACCACAACAA-loop-TTGTGTG.

The multicistronic construct mentioned above is also disclosed, in which each loop comprises 9 nucleotides.

In addition, the multicistronic construct disclosed above is disclosed, in which each loop comprises 9 nucleotides, and in which each loop is ATATATAAT.

The multicistronic construct mentioned above is also disclosed which comprises at least a first and a second autocomplementary sequences, in which the construct is used to inhibit the formation of tumors and to reverse previously formed tumors previously disclosed, and in which the autocomplementary sequences they are separated by an intervention sequence with a length of approximately 22-35 base pairs.

In addition, the multicistronic construct disclosed above is disclosed, in which the intervention sequence is GATCCA CTAGTAACGG CCGCCAGTGT GCTGG AATT.

The multicistronic construct mentioned above is also disclosed which comprises at least a first and a second autocomplementary sequences, in which the construct is used to inhibit the formation of tumors and

Claims (12)

image 1 1. A bicistronic short interference RNA construct comprising a first autocomplementary urokinase plasminogen activator receptor (uPAR) sequence and a second sequence 5 autocomplementary type 9 matrix metalloprotease (MMP-9) and an intervention sequence between the first and second autocomplementary sequences of approximately 22-68 base pairs. 2. The bicistronic construct of claim 1, wherein the autocomplementary sequence of uPAR is CTACAGCAGTGGAGAGCGATT-loop-AATCGCTCTCCACTGCTGTAG (SEQ ID NO: 2) and the sequence 10 autocomplementary MMP-9 is CAAGTGGCACCACCACAACAA-loop-TTGTTGTGGTGGTGCCACTTG (SEQ ID NO: 4). 3. The bicistronic construct of claim 2, wherein each loop comprises 9 nucleotides.

The bicistronic construct of claim 3, wherein each loop is ATATATAAT.

5. The bicistronic construct of claim 1, wherein the intervention sequence is GATCCA CTAGTAACGG CCGCCAGTGT GCTGG AATT (SEQ ID NO: 5).

The bicistronic construct of claim 1, wherein the construct is a circular nucleic acid.

7. Pharmaceutical composition comprising the short interference RNA bicistronic construct of any one of claims 1 to 6.

The pharmaceutical composition of claim 7, wherein the bicistronic short interference RNA construct is directed to the urokinase type plasminogen activator (uPAR) receptor and to the type 9 matrix metalloprotease (MMP-9).

9. The pharmaceutical composition of claim 7, wherein the bicistronic interference RNA construct Short 30 comprises CTACAGCAGTGGAGAGCGATT-loop-AATCGCTCTCCACTGCTGTAG-intervention sequence-CAAGTGGCACCAC-CACAACAA-loop-TTGTTGTGGTGGTGCCACTTG (SEQ ID NO: 2 and 4 respectively in order of appearance). 10. The bicistronic construct of any one of claims 1-6 for inhibiting tumor formation and reversing previously formed tumors. 11. The bicistronic construct for the use of claim 10, wherein the tumors are inhibited by reducing at least one of tumor cell proliferation, tumor cell invasion, tumor cell migration and angiogenesis. 40 12. The bicistronic construct for the use of claim 10, wherein the tumors are selected from the group consisting of prostate cancer, glioma, breast cancer and melanoma. 13. The bicistronic construct for the use of claim 12, wherein the construct must be administered systemically. 14. The bicistronic construct for the use of claim 12, wherein the construct must be administered through direct administration. 15. A recombinant cell transformed with the bicistronic construct of claim 1. 16. A recombinant virus transformed with the bicistronic construct of claim 1. 49